Regulators of type-1tumor necrosis factor receptor and other cytokine receptor shedding

ABSTRACT

The present invention provides compositions and methods for the regulation of cytokine signaling through the Tumor Necrosis Factor (TNF) pathway. Specifically, the invention provides a novel gene, polypeptide and related compositions and methods for the regulation of ectodomain shedding. In preferred embodiments, methods and compositions for the regulation of TNF Type-1 Receptor ectodomain shedding are provided. The present invention finds use in therapeutics, diagnostics, and drug screening applications.

[0001] This application claims priority benefit to U.S. ProvisionalPatent Application No. 60/185,586, filed Feb. 28, 2000.

[0002] This invention was made during the course of work supported bythe United States Government under the National Institutes of Health. Assuch, the United States Government may have certain rights to thisinvention.

FIELD OF THE INVETION

[0003] The present invention provides compositions and methods relatedto regulation of cytokine signaling through the Tumor Necrosis Factor(TNF) pathway. Specifically, the invention provides novel genes,polypeptides and related compositions and methods for the regulation ofTNF Type-1 Receptor ectodomain shedding. It is contemplated that thecompositions and methods of the present invention will also find use inthe regulation of ectodomain shedding of other cytokine receptors. It isfurther contemplated that these compositions and methods will find usein therapeutics for the treatment of diseases and disorders of theimmune system.

BACKGROUND OF THE INVENTION

[0004] Aberrant regulation of cytokine signaling results in a widevariety of hyper-inflammatory, autoimmune and immune-deficiencypathological conditions. Cytokines are a large and diverse group ofmolecules which mediate interactions between cells, ultimatelyregulating the wide variety of cells of the immune repertoire. Cytokinesignaling mediates numerous facets of normal immune system physiology,including development, response, activation, maintenance, memory andapoptosis (Roitt et al. (Eds.), Immunology, Fifth Edition, MosbyInternational Publishers [1998]).

[0005] Tumor Necrosis Factor

[0006] Tumor necrosis factor-α (commonly written as TNF, but alsowritten as tumor necrosis factor or TNFα) is a multifunctional cytokinemediating pleiotropic biological functions in both health and diseasestates. TNF is secreted primarily by monocytes and macrophages, but canalso be secreted by other cell types. The list of processes regulated byTNF is extensive, and includes inflammation, immunoregulation,cytotoxicity and antiviral effects (See e.g., Vilcek et al, J. Biol.Chem., 266:7313-7316 [1991]). TNF plays an integral role in destroyingtumors, mediating responses to tissue injury, and protecting hosts frominfections by various microorganisms (Vassali, Ann. Rev. Immunol.,10:411-452 [1992]). TNF also induces the transcriptional activation ofnumerous genes, including NF-κB and AP-1, with the consequent expressionof pro-inflammatory and immunoregulatory genes (Rothe et al., Cell83:1243-124 [1995]; Varfolomeev et al., J. Exp. Med., 183:1271-1275[1996]; Chinnaiyan et al., J. Biol. Chem., 271:4961-4965 [1996]; Hsu etal., Immunity 4:387-396 [1996]; and Hsu et al., Cell 84:299-308 [1996]).TNF-mediated NF-κB activation is also an important negative feedbackmechanism regulating apoptosis (Beg and Baltimore, Science 274:782-784[1996]; Van Antwerp et al., Science 274:787-789 [1996]; and Wang et al.,Science 274:784-787 [1996]).

[0007] TNF in Disease

[0008] TNF has also been implicated in the pathogenesis of a variety ofdiseases and disorders. It is theorized that these pathologies resultfrom the aberrant regulation of TNF activity, in which the pathologiesmanifest as a result of excessive or insufficient TNF activity. Amongthe activities for which TNF is most noted are its pro-inflammatoryactions, sometimes termed the “acute phase immune response.”Unfortunately, if not properly regulated, these proinflammatoryresponses can result in tissue injury and chronic inflammatory diseases,such as rheumatoid arthritis, inflammatory bowel disease, septic shock,cachexia, autoirumune disorders, graft-versus-host disease and insulinresistance (Piguet et al., J. Exp. Med., 166:1280 [1987]; Pujol-Borrellet al., Nature 326:304-306 [1987]; Tracey et al., Nature 330:662-664[1987]; Oliff, Cell 54:141-142 [1988]; Vilcek et al., J. Biol. Chem.,266:7313-7316 [1991]; and Eigler et al., Immunol. Today 18:487-92[1997]). Excessive TNF activity results in the detrimental effects of anexaggerated immune response demonstrated in some of these diseases,exemplified by overstimulation of interleukin-6 andgranulocyte/macrophage-colony stimulating factor (GM-CSF) secretion,enhanced cytotoxicity of polymorphonuclear neutrophils, prolongedexpression of cellular adhesion molecules, induction of procoagulantactivity on vascular endothelial cells, increased adherence ofneutrophils and lymphocytes, and stimulation of the release of plateletactivating factor from macrophages, neutrophils and vascular endothelialcells (Vassali, Ann. Rev. Immunol., 10:411-452 [1992]; Vilcek et al., J.Biol. Chem., 266:7313-7316 [1992]; and Barbara et al., Immunol. and CellBiol., 74:434-443 [1996]).

[0009] Recent evidence also implicates TNF activity in the pathogenesisof many infections. TNF is thought to play a central role in thepathophysiological consequences of Gram-negative sepsis and endotoxicshock, including fever, malaise, anorexia, and cachexia (Beutler et al.,Nature 316:552-554 [1985]; Bauss et al, Infect. Immun., 55:1622-1625[1987]; Tracey et al., Nature 330:662-664 [1987]; and Vassali, Ann. Rev.Immunol., 10:411-452 [1992]). Because TNF can mimic many of thebiological effects of endotoxin, it is theorized that TNF is a centralmediator responsible for the clinical manifestations ofendotoxin-related and other critical illnesses (Waage et al., Lancet1:355-357 [1987]; Cerami et al., Immunol. Today 9:28 [1988]; Mitchie etal., N. Eng. J. Med., 318:1481-1486 [1988]; Revhaug et al., Arch. Surg.,123:162-170 [1988]; and Michie et al., Ann. Surg., 209:19-24 [1989]).

[0010] Tumor Necrosis Factor Receptor

[0011] The numerous biological effects of TNF are now know to bemediated by two transmembrane receptors, the 55 kilodalton Type Ireceptor (also written as “CD120a,” and referred to herein as “TNFR1”)and the 75 kilodalton Type II receptor (also written as “CD120b,” andreferred to herein as TNFR2). Although both TNFR1 and TNFR2 demonstratestrong affinity for TNFα, these two receptors demonstrate no apparenthomology in their cytoplasmic (i.e., intracellular) domains. This factis consistent with the observation that these two receptors transducedifferent signals to the nucleus via distinct signaling intermediates(Lewis et al., Proc. Natl. Acad. Sci. USA 88:2830-2834 [1991]; Tartagliaand Goeddel, Immunol. Today 13:151-153 [1992]; and Barbara et al.,Imunol. Cell Biol., 74:434-443 [1996]).

[0012] Soluble TNF inhibitors have been identified in normal humanurine, as well as in sera and other body fluids of patients withinfectious, neoplastic and immunologic disorders. This observationultimately led to the revelation that these soluble TNF inhibitors wereactually the extracellular domains of TNF receptors derived byproteolytic cleavage of the transmembrane forms (Engelmann et al., J.Biol. Chem., 264:11974-11980 [1989]; Olsson et al., Eur. J. Haematol.,42:270-275 [1989]; Seckinger et al., J Biol. Chem., 264:11966-11973[1989]; Engelmann et al., J. Biol. Chem., 265:1531-1536 [1990]; andAderka et al., J. Exp. Med., 175:323-329 [1992]). In the case of theTNFR1, this proteolytic activity results in the cleavage and shedding ofthe extracellular N-terminal domain (also called the ectodomain). Thesefree, soluble TNFR1 ectodomains (“sTNFR1s”) have an affinity for TNFthat is similar to that of intact membrane receptors. Due to thisaffinity, the free receptors are able to bind and sequester TNF, therebyinhibiting the biological action of TNF. Furthermore, the generation ofsTNFR1 is also likely to suppress TNF signaling by reducing the numberof functional TNF receptors acting at the cell membrane. The sTNFR1ectodomains are also theorized to serve a more complex bufferingfunction in the regulation of TNF activity (Aderka et al., J. Exp. Med.,175:323-329 [1992]; and Werb and Yan, Science 282:1279-1280 [1998]). Thecomplexity of TNF signaling is fuirther illustrated by the observationthat many of the stimuli that result in TNF release also result in therelease of the soluble TNF receptor, suggesting that these soluble TNFinhibitors may serve as part of a regulated feedback mechanism tocontrol TNF activity (Adreke et al., J. Exp. Med., 175:323-329 [1992];and Porteu and Nathan, J. Exp. Med., 172:599-607 [1990]).

[0013] The importance of TNFR1 in the regulation of TNF activity in hostdefense, immunoregulation and development has been fuirther demonstratedin studies utilizing TNFR1 knockout mice. Mice deficient in TNFR1 show avariety of phenotypes, including phenotypes which mimic human immunedisorders (Pfeffer et al., Cell 73:457-467 [1993]; Rothe, Nature364:798-802 [1993]; Le Hir et al., J. Exp. Med., 183:2367-2372 [1996];Matsumoto et al., Science 271:1289-1291[1996]; Mori et al. J. Immunol.157:3178-3182 [1996]; Speiser et al., J. Immunol., 158:5185-5190 [1997];Tkachuk et al., J. Exp. Med., 187:469-477 [1998]; and Kagi et al., J.Immunol., 162:4598-4605 [1999]).

[0014] The key role of TNFR1 shedding in the regulation of TNFbioactivity is highlighted by the association of germline mutations inTNFR1 extracellular domains with impaired TNFR1 shedding andautoinflammatory disease characterized by autosomal dominant periodicfever syndromes (McDermott et al., Cell 97:133-144 [1999]).

[0015] Other Mediators of Acute Phase Response

[0016] In addition to TNF, other cytokines have been implicated in theinduction of the pro-inflammatory response (i.e., the acute phase immuneresponse). These cytokines which demonstrate overlapping activities withTNF include the interleukins (e.g., IL-1 and IL-6) (Suffredini et al.,J. Clin. Immunol., 19:203-214 [1999]).

[0017] IL-1 (consisting of both α and β forms) is an importantproinflammatory cytokine which regulates the expression of a widevariety of target genes and proteins in nearly every cell type(Dinarello, Blood 77:1627-1652 [1991]; and Dinarello, The CytokineHandbook (ed. Angus W. Thomson), 3^(rd) edition, Academic Press, SanDiego, p. 35-72 [1998]). The spectrum of IL-1-mediated biologic effectsincludes inflammatory, metabolic, physiologic, hematopoietic, andimmunologic functions. IL-1 is thought to play a role in thepathogenesis of several disease states, including septic shock,rheumatoid arthritis, inflammatory bowel disease, myelogenous leukemia,diabetes mellitus, and atherosclerosis (Dinarello et al., N. Engl. J.Med., 328:106-113 [1993]).

[0018] IL-6 is also a multifunctional cytokine with pleiotropicpro-inflammatory effects (DiCosmo, et al., J. Clin. Invest.,94:2028-2035 [1994]; and Kishimoto et al., Blood 86:1243-1254 [1995]).For example, IL-6 plays an important role in regulating B cellimmunoglobulin production, T-cell activation, growth anddifferentiation, hematopoiesis, hepatic acute phase reactions andosteoclast development (Hirano, The Cytokine Handbook (ed. Angus W.Thomson), 3^(rd) edition, Academic Press, San Diego, p. 197-228 [1998]).Dysregulated production of IL-6 may contribute to the pathogenesis of avariety of inflammatory, neoplastic and autoimmune disorders, such asplasma cell neoplasia and Castleman's disease (Yoshizaki, et al., Blood74:1360-1367 [1989]; and Hirano, Int. J. Cell Cloning 9:166-184 [1991]).

[0019] The signal transduction pathways utilized by TNF, IL-1 and IL-6also show shared signaling intermediates. For example, both TNF and IL-1can activate both NF-κB and AP-1, which are important pro-inflammatorytranscription factors (Ashkenazi et al., Science 281:1305-1308 [1998];and Dinarello, The Cytokine Handbook (ed. Angus W. Thomson), 3^(rd)edition, Academic Press, San Diego, p. 35-72 [1998]). Similarly, IL-6signaling uses components of the JAK-STAT pathway, which has also beenreported to be induced by TNF (Guo et al., J. Immunol., 160:2742-2750[1998]; and Hirano, The Cytokine Handbook (ed. Angus W. Thomson), 3^(rd)edition, Academic Press, San Diego, p. 197-228 [1998]).

[0020] The cognate receptors for the IL-1 and IL-6 cytokines are known.There are two IL-1 receptor forms, type I and type II. There is a singleIL-6 receptor, consisting of gp80 alpha chain and gp130 beta chainsubunits, where ligand binding is mediated by the alpha subunit. TheIL-1 and IL-6 receptors are also present as soluble forms analogous tothe soluble form of TNFR1. Furthermore, it has been suggested that thesereceptors play a role in the regulation of IL-1 and IL-6 activity andpro-inflammatory response (Dower et al., J. Immunol., 142:4314 [1989];Novick et al., J. Exp. Med., 170:1409 [1989]; Eastgate et al., FEBSLett., 260:213 [1990]; Giri et al., J. Biol. Chem., 265:17416 [1990];Symons et al., Cytokine 2:190 [1990]; Symons et al., FEBS Lett., 272:133[1990]; Symons et al., J. Exp. Med., 174:1251-1254 [1991]; Mullberg etal., Biochem. Biophys. Res. Commun., 189:794 [1992]; Mullberg et al.,Eur. J. Immunol., 23:473 [1993]; Svenson et al., Cytokine 5:427 [1993];and Arend et al., J. Immunol., 153:4766-4774 [1994]).

[0021] Analogy between regulation of TNF and other cytokines is furtherillustrated by studies utilizing peptide-hydroxamate metalloproteaseinhibitors. Specifically, the protease inhibitors TAPI (TNF-α proteaseinhibitor) and RU36156 have been reported to inhibit the proteolyticcleavage and shedding of both TNFR1 and IL-6R (Mullberg et al., J.Immunol., 155:5198-5205 [1995]; and Gallea-Robache et al., Cytokine9:340-346 [1997]).

[0022] As discussed above, in view of the importance of TNF, IL-1 andIL-6 in both health and disease states, there exists a need for methodsand compositions for the regulation of TNF, IL-1 and IL-6 cytokineactivity. These methods and compositions will find use as therapeuticagents for the treatment of disease states.

SUMMARY OF THE INVENTION

[0023] The present invention provides compositions and methods relatedto regulation of cytokine signaling through the Tumor Necrosis Factor(TNF) pathway, as well as other signaling pathways controlled by othercytokines, including IL-1 and IL-6. It is contemplated that thesecompositions and methods will find use in therapeutics for the treatmentof diseases and disorders of the immune system.

[0024] The present invention also provides novel polypeptides and anucleic acid sequences. In particular, the present invention providesisolated nucleic acids comprising the nucleotide sequence set forth inSEQ ID NO:1, which encodes a polypeptide referred to as “ARTS-”1 (i.e.,aminopeptidase regulator of type I, 55 kDa tumor necrosis factorreceptor ectodomain shedding) which has the ability to promote theshedding of the extracellular domain of Type I Tumor Necrosis FactorReceptor (TNFR1). Thus, the present invention also provides the isolatedpolypeptides comprising the amino acid sequence set forth in SEQ IDNO:2, as well as isolated nucleic acids encoding the polypeptide setforth in SEQ ID NO:2.

[0025] The present invention further provides recombinant vectorscomprising the ARTS-1 gene and host cells comprising these vectors. Inone embodiment, the host cell is eukaryotic, while in an alternativeembodiment, the host cell is prokaryotic. The present invention alsoprovides antibodies raised against at least a portion of the ARTS-1polypeptide of SEQ ID NO:2. In some embodiments, the antibodies aremonoclonal, while in alternative embodiments, the antibodies arepolyclonal.

[0026] In other embodiments, the present invention provides isolatednucleic acids that are substantially homologous to the nucleic acid ofSEQ ID NO:1, wherein the nucleic acid is capable of hybridizing underhigh stringency conditions to the nucleic acid of SEQ ID NO:1. In apreferred embodiment, the nucleic acid substantially homologous to theARTS-1 gene encodes a polypeptide having the ability to regulate theshedding of the extracellular domain of at least one cytokine receptor.In particularly preferred embodiments, the cytokine receptor is selectedfrom the group consisting of type-1 tumor necrosis factor receptor, typeI interleukin-1 cytokine receptor, type II interleukin-1 cytokinereceptor, and interleukin-6 cytokine receptor alpha-chain gp80. In someembodiments, the nucleic acid substantially homologous to the nucleicacid of the ARTS-1 gene is identified using PCR methods. In alternativeembodiments, the nucleic acid substantially homologous to the ARTS-1gene is identified using hybridization screening methods.

[0027] The present invention further provides methods for the isolationof amplifiable nucleic acid substantially homologous to the nucleic acidof SEQ ID NO:1 comprising: providing a sample comprising templatenucleic acid suspected of encoding a gene substantially homologous tothe nucleic acid of SEQ ID NO:1; and at least two primers; annealing theprimers to the template nucleic acid; extending the primers (e.g., withreiterated DNA synthesis) under conditions such that the templatenucleic acid is amplified, to produce an amplified product; andvisualizing the amplified product. In some preferred embodiments, theamplified product is isolated. The present invention further providesthe product of these amplification methods. In preferred embodiments,the amplified product encodes a polypeptide having the ability toregulate the shedding of the extracellular domain of at least onecytokine receptor. In particularly preferred embodiments, the cytokinereceptor is selected from the group consisting of type-1 tumor necrosisfactor receptor, type I interleukin-1 cytokine receptor, type IIinterleukin-1 cytokine receptor, and interleukin-6 cytokine receptoralpha-chain gp80.

[0028] The present invention also provides methods for the use of thesecompositions to regulate the shedding of sTNFR1. It is contemplated thatmethods which regulate the shedding of the sTNFR1 also regulate theactivity of TNF. In a most preferred embodiment, the invention providesmethods for use of these compositions in therapeutic applications in thetreatment of immune system diseases and disorders resulting fromaberrant cytokine activity.

[0029] The present invention provides methods for regulating theshedding of the extracellular domain of at least one cytokine receptor,comprising the steps of: providing a recombinant vector comprising SEQID NO:1 in the sense orientation, a first tissue containing one or morecells expressing at least one cytokine receptor, and a second tissuecomprising one or more cells capable of expressing the polypeptideencoded by the recombinant vector; delivering the vector to the cells ofthe second tissue in the presence of the first tissue, under conditionswhich result in regulation of shedding of the cytokine receptor(s) fromcells of the first tissue. In some preferred embodiments, the cytokinereceptor is selected from the group consisting of type-1 tumor necrosisfactor receptor, type I interleukin-1 cytokine receptor, type IIinterleukin-1 cytokine receptor, and interleukin-6 cytokine receptoralpha-chain gp80. In alternative preferred embodiments, the delivery ofthe vector to the second tissue comprises a means of intracellulardelivery selected from the group consisting of direct nucleic acidadministration, liposome administration, viral vector delivery, and exvivo gene delivery followed by transplantation.

[0030] In other embodiments, the present invention provides compositionsand methods suitable for regulating TNFR1 ectodomain shedding byoverexpressing or suppressing the activity of the ARTS-1 polypeptide. Insome of these embodiments, TNFR1 ectodomain shedding is regulated by theintracellular delivery of a vector which results in overexpression ofthe ARTS-1 polypeptide (e.g., SEQ ID NO:2). In another embodiment, TNFR1ectodomain shedding is regulated by delivering purified ARTS-1polypeptide (e.g., SEQ ID NO:2) to tissues.

[0031] The present invention also provides methods for regulating theshedding of the extracellular domain of at least one cytokine receptor,comprising the steps of: providing a recombinant vector comprising atleast a transcribeable portion of the nucleic acid of SEQ ID NO:1 in anantisense orientation, a first tissue comprising one or more cellsexpressing at least one cytokine receptor, and a second tissuecomprising one or more cells expressing the endogenous polypeptide ofSEQ ID NO:2, and one or more cells capable of transcribing the antisensenucleic acid; and delivering the vector to the second tissue in thepresence of the first tissue, under conditions that result in regulationof shedding of the cytokine receptor(s) from the cells of the firsttissue. In preferred embodiments, the cytokine receptor is selected fromthe group consisting of type-1 tumor necrosis factor receptor, type Iinterleukin-1 cytokine receptor, type II interleukin-1 cytokinereceptor, and interleukin-6 cytokine receptor alpha-chain gp80. Inalternative preferred embodiments, the delivery of the vector to thesecond tissue comprises a means of intracellular delivery selected fromthe group consisting of direct nucleic acid administration, liposomeadministration, viral vector delivery, and ex vivo gene deliveryfollowed by transplantation.

[0032] The present invention further provides methods for regulating theshedding of the extracellular domain of at least one cytokine receptor,comprising the steps of: providing a polypeptide having the amino acidsequence set forth in SEQ ID NO:2, and a tissue comprising one or morecells expressing at least one cytokine receptor on their plasma membraneextracellular surface; and delivering the polypeptide to the tissueunder conditions such that the polypeptide regulates the shedding of thecytokine receptor(s) from the surface of the cells of the tissue. Insome preferred embodiments, the cytokine receptor is selected from thegroup consisting of type-1 tumor necrosis factor receptor, type Iinterleukin-1 cytokine receptor, type II interleukin-1 cytokinereceptor, and interleukin-6 cytokine receptor alpha-chain gp80. Inalternative preferred embodiments, the delivery of the polypeptide tothe tissue comprises a means of delivery selected from the groupconsisting of oral administration, intra-arterial injection, intravenousinjection, intramuscular injection, intraperitoneal injection,subcutaneous injection, suppository, local surgical administration,systemic surgical administration, catheter, and any combination of thesemeans of delivery.

[0033] The present invention further provides methods for regulating theshedding of the extracellular domain of at least one cytokine receptor,providing an antibody raised against the ARTS-1 polypeptide, and atissue comprising one or more cells expressing at least one cytokinereceptor and the endogenous polypeptide of SEQ ID NO:2; and deliveringthe antibody to the tissue under conditions such that the antibodyregulates the shedding of the cytokine receptor(s) from the surface ofthe cells of the tissue. In some preferred embodiments, the cytokinereceptor is selected from the group consisting of type-1 tumor necrosisfactor receptor, type I interleukin-1 cytokine receptor, type IIinterleukin-1 cytokine receptor, and interleukin-6 cytokine receptoralpha-chain gp80. In alternative preferred embodiments, the means ofdelivery is selected from the group consisting of oral administration,intra-arterial injection, intravenous injection, intramuscularinjection, intraperitoneal injection, subcutaneous injection,suppository, local surgical administration, systemic surgicaladministration, catheter, and any combination of these means ofdelivery.

[0034] In view of the overlapping activities between TNF and otherproinflammatory cytokines, the present invention also providescompositions and methods suitable for regulating the shedding of othercytokine receptors in addition to TNFR1, including, but not limited toIL-1 and IL-6 cytokine receptors.

[0035] In some particularly preferred embodiments, the present inventionprovides compositions and methods are provided to treat subjectsdisplaying pathology, as well as subjects suspected of displaying or atrisk of displaying pathology resulting from abnormal cytokine activity.The compositions provided for use in the most preferred embodimentinclude vectors capable of expressing the ARTS-1 polypeptide, vectorscapable of transcribing at least a portion of the ARTS-1 gene in anantisense orientation, the ARTS-1 polypeptide set forth in SEQ ID NO:2,and antibodies raised against at least a portion of the ARTS-1polypeptide.

[0036] The present invention also provides methods for treating asubject, comprising the steps of: providing a composition selected fromthe group consisting of a recombinant vector comprising at least aportion of SEQ ID NO:1 in the sense orientation, a recombinant vectorcomprising at least a portion of SEQ ID NO:1 in the antisenseorientation, at least a portion of the ARTS-1 polypeptide, at least aportion of SEQ ID NO:2, and antibody directed against at least a portionof the ARTS-1 polypeptide, as well as a subject, and a means of deliveryof the composition to at least one tissue of the subject; and deliveringthe composition to the subject using the means of delivery. In preferredembodiments, the subject is selected from the group consisting of asubject displaying pathology resulting from abnormal cytokine activity,a subject suspected of displaying pathology resulting from abnormalcytokine activity, and a subject at risk of displaying pathologyresulting from abnormal cytokine activity. In some preferredembodiments, the cytokine activity is mediated by a cytokine selectedfrom the group consisting of tumor necrosis factor α, interleukin-1alpha, interleukin-1 beta, and interleukin-6. In some particularlypreferred embodiments, the subject is a human. In alternative preferredembodiments, the means of delivery is selected from the group consistingof oral administration, intra-arterial injection, intravenous injection,intramuscular injection, intraperitoneal injection, subcutaneousinjection, suppository, local surgical administration, systemic surgicaladministration, catheter, and any combination of these means ofdelivery. In further preferred embodiments, the means of delivery isfurther selected from the group consisting of direct nucleic acidadministration, liposome administration, viral vector delivery, and exvivo gene delivery followed by transplantation.

[0037] In other embodiments, the present invention provides ARTS-1markers, including those selected from the group consisting of theARTS-1 mRNA transcript and the ARTS-1 polypeptide. Furthermore, theinvention also provides compositions and means for detecting the ARTS-1mRNA and polypeptide markers. In preferred embodiments, thesecompositions are selected from the group consisting of nucleic acidcomplementary to the ARTS-1 mRNA, and antibodies specific for at least aportion of the ARTS-1 polypeptide.

[0038] The present invention also provides means for detecting an ARTS-1mRNA in a sample, wherein the means comprises at least a portion of thenucleic acid of SEQ ID NO:1 complementary to at least a portion ofARTS-1 mRNA, and further wherein the nucleic acid is a probe. Inalternative embodiments, the means comprises Northern blotting. Inpreferred embodiments, the sample is a tissue sample from a subject. Inadditional preferred embodiments, the methods provide means fordetecting an ARTS-1 polypeptide in a sample, wherein the means comprisesan antibody directed against at least a portion of the ARTS-1polypeptide. In some preferred embodiments, the means comprises Westernimmunoblotting, while in alternative preferred embodiments, the meanscomprises an enzyme-linked immunosorbent assay. In alternative preferredembodiments, the sample is a tissue sample from a subject.

[0039] The present invention also provides diagnostic kits comprising ameans to measure ARTS-1 expression, wherein the means comprises at leasta portion of SEQ ID NO:1 that is complementary to at least a portion ofARTS-1 mRNA, and further wherein the nucleic acid is a probe. Inalternative embodiments, the diagnostic kits of the present inventionprovides means for detecting an ARTS-1 polypeptide in a sample, whereinthe means comprises antibody directed against at least a portion of theARTS-1 polypeptide. In some preferred embodiments, the means comprisesWestern immunoblotting, while in alternative preferred embodiments, themeans comprises an enzyme-linked immunosorbent assay.

[0040] The present invention further provides compositions and methodsfor the screening for drugs with the ability to regulate ARTS-1peptidase activity and cytokine receptor shedding regulatory activity.In some embodiments, the present invention provides methods for drugscreening to identify drugs having the ability to regulate ARTS-1expression comprising the steps of: providing a drug, cultured cells,and a means to measure ARTS-1 expression, wherein the means comprises atleast a portion of SEQ ID NO:1 that is complementary to at least aportion of ARTS-1 mRNA, and further wherein the nucleic acid is a probe;exposing the cells to the drug; and using the means to measure ARTS-1expression. In alternative embodiments, the diagnostic kits of thepresent invention provide means for detecting an ARTS-1 polypeptide in asample, wherein the means comprises an antibody directed against atleast a portion of the ARTS-1 polypeptide. In some preferredembodiments, the cultured cells are human NCI-H292 pulmonarymucoepidermoid carcinoma cells.

[0041] The present invention further provides methods for drug screeningto identify drugs capable of regulating the peptidase activity ofARTS-1, comprising the steps of: providing purified ARTS-1 polypeptide,an amino acid p-nitroaniline, a means to measure amino acidp-nitroaniline cleavage, and a drug; exposing the purified ARTS-1polypeptide to the amino acid p-nitroaniline in the absence and presenceof the drug; and measuring amino acid p-nitroaniline cleavage in theabsence and presence of the drug. In some preferred embodiments, thepurified ARTS-1 polypeptide comprises glutathione-S-transferase. Inalternative preferred embodiments, the amino acid p-nitroaniline isselected from the group consisting of isoleucine p-nitroanilide,phenylalanine p-nitroanilide and glycine p-nitroanilide. In stillfurther preferred embodiments, the means to measure amino acidp-nitroaniline cleavage comprises measuring absorbance at 380 nm.

[0042] The present invention also provides methods for drug screening toidentify drugs capable of regulating the shedding of a cytokinereceptor, comprising the steps of: providing cultured cells expressingat least one cytokine receptor, a means to quantitate the concentrationof the soluble form of the cytokine receptor(s) in the supernatants ofthe cultured cells, and a drug; culturing the cells in the absence andpresence of the drug; quantitating the concentration of the soluble formof the cytokine receptor(s) in the supernatants of the cultured cells;and comparing the concentrations of the soluble cytokine receptor(s) inthe supernatants of the cell cultures in the absence and presence ofdrug. In some preferred embodiments, the cytokine receptor is selectedfrom the group consisting of type-1 tumor necrosis factor receptor, typeII interleukin-1 cytokine receptor, and interleukin-6 cytokine receptoralpha-chain gp80. In alternative preferred embodiments, the means toquantitate the concentration of the soluble form of a cytokine receptorcomprises an enzyme-linked immunosorbent assay. In further embodiments,the cultured cells are cultured human NCI-H292 pulmonary mucoepidermoidcarcinoma cells.

DESCRIPTION OF THE FIGURES

[0043]FIG. 1 presents the ARTS-1 transcription unit and open readingframe translation.

[0044]FIG. 2 provides a schematic representation of the ARTS-1 protein,indicating domains of homology with the aminopeptidase family ofgluzincin zinc metalloproteases.

[0045]FIG. 3 provides a Northern blot analysis of multiple human tissuesusing a ³²p-labelled ARTS-1 cDNA probe. Panel A shows the blot probedwith the ARTS-1 cDNA probe. Panel B shows the same blot followingstripping and rehybridization to a probe specific for the humanglyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a reference forRNA loading normalization.

[0046]FIG. 4 provides Western immunoblots using pre-immune andpolyclonal anti-ARTS-1 antisera in conjunction with crude whole cellhomogenates, and membrane and cytosolic fractions made from culturedNCI-H292 cells. Panel A provides a Western immunoblot using the immunesera. Panel B provides a Western immunoblot using the pre-immune sera.Panels C and D provide results from Western immunoblot competitionexperiments using bovine serum albumin or ARTS-1 cognate peptide,respectively.

[0047]FIG. 5 provides Western immunoblots using polyclonal anti-ARTS-1antisera and membrane and cytosolic fractions made from primary cellsand cell lines. Panel A provides a Western immunoblot using humanbronchial brush cells collected from human subjects. Panel B provides aWestern immunoblot using the NCI-H292, BEAS-2B, BET-1A and A549 culturedcell lines. Panel C provides a Western immunoblot using primary culturesof normal human bronchial epithelial cells (NHBE), human umbilical veinendothelial cells (HUVEC) and human fibroblasts.

[0048]FIG. 6 provides results of an analysis of recombinant GST-ARTS-1fusion protein purification and analysis of the aminopeptidase activityof this protein following purification. Panel A provides a Coomassiestained gel of protein samples obtained during the production andpurification of the GST-ARTS-1 fusion protein. Panel B provides an FPLCelution profile of the purified GST-ARTS-1 fusion protein. Panel Cprovides the results of an assay of phenylalanine p-nitroanilinesubstrate aminopeptidase activity of the FPLC eluted fractions.

[0049]FIG. 7 provides a Western immunoblot using anti-ARTS-1 polyclonalantiserum as the primary antibody. Samples analyzed in the blot are frommembrane protein fractions derived from stably transfected NCI-H292cells which were untransfected (WT), control transfected (Mock), ARTS-1sense-overexpressing (ARTS-1) or ARTS-1 anti-sense expressing (AS). Twoindependent clones each from the ARTS-1 and AS cell lines were analyzed.

[0050]FIG. 8 provides results of an ELISA to determine the levels ofsTNFR1 resulting from TNFR1 ectodomain shedding in cell culturesupernatants from cultures of the same cell lines as indicated for FIG.7.

[0051]FIG. 9 provides a graph depicting the ability of ARTS-1overexpression to potentiate the cleavage and shedding of TNFRectodomain from the surface of NCI-H292 cells in response to PMAstimulation, as determined by an ELISA measuring sTNFR1 in the cellculture supernatants.

[0052]FIG. 10 provides a histogram showing the results of an ELISAanalysis. The ELISA determined the levels of sTNFR1 (a measure of TNFR1ectodomain shedding) in cell culture supernatants for stably transfectedNCI-H292 cell lines expressing various ARTS-1 mutants.

[0053]FIG. 11 provides a Western immunoblot using an anti-TNFR1 antibodyas the primary antibody to detect membrane bound TNFR1. Samples analyzedin the blot include membrane protein fractions derived from stablytransfected NCI-H292 cells which were untransfected (WT), controltransfected (Mock), ARTS-1 sense-overexpressing (ARTS-1), or ARTS-1anti-sense expressing (AS). Two independent clones of each cell linewere analyzed.

[0054]FIG. 12 provides two Western immunoblots following two in vivoimmunoprecipitation experiments using membrane protein fractionsisolated from cultured NCI-H292 cells. In the top panel, an anti-TNFR1antibody was used in the immunoprecipitation step (indicated as “IP”),and anti-ARTS-1 antiserum was used as the primary antibody in theimmunoblotting (indicated as “IB”). Conversely, in the lower panel, theanti-ARTS-1 antiserum was used in the immunoprecipitation, while theanti-TNFR1 antibody was used as the primary antibody in theimmunoblotting.

[0055]FIG. 13 provides a Western immunoblot following an in vivoimmunoprecipitation experiment using an anti-TNFR1 monoclonal antibodyfor the immunoprecipitation and anti-ARTS-1 antiserum as the primaryantibody in the blot. The immunoprecipitations used cell membraneprotein fractions derived from stably transfected NCI-H292 cellsoverexpressing ARTS-1 (ARTS-1), expressing an anti-sense ARTS-1 message(AS), as well as control-transfected (Mock) and non-transfected (WT)cell lines.

[0056]FIG. 14 provides results of two drug screening assays. Panel Aprovides a Western immunoblot using anti-ARTS-1 polyclonal antiserum asthe primary antibody, tested with membrane protein fractions fromNCI-H292 cells following exposure to 4b-phorbol 12-myristate 13-acetate(PMA) (over a time course). Panel B provides the results of an ELISA todetermine the levels of sTNFR1 in cell culture supernatants fromNCI-H292 cell cultures following exposure PMA, over time.

DEFINITIONS

[0057] To facilitate an understanding of the present invention, a numberof terms and phrases are defined or clarified below:

[0058] The terms “peptide,” “polypeptide” and “protein” all refer to aprimary sequence of amino acids that are joined by covalent “peptidelinkages.” In general, a peptide consists of a few amino acids,typically from 2-25 amino acids, and is shorter than a protein.Polypeptides may encompass either peptides or proteins. Where “aminoacid sequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms, such as “polypeptide” or “protein” are not meant to limit theamino acid sequence to the complete, native amino acid sequenceassociated with the recited protein molecule.

[0059] As used herein, the term “nucleic acid” refers to any sequence ofthe bases adenine, thymine, cytosine and guanine, and various analogs ofthese bases. A nucleic acid is characterized by a specific nucleotidesequence (i.e., the sequence of the bases and base analogs in themolecule). A “nucleic acid” is not limited to DNA or RNA, and is notlimited in any way by the size of the molecule. A nucleic acid may bedouble stranded or single stranded. The term “nucleic acid” encompassessequences that include any of the bases adenine, thymine, guanine andcytosine, as well as known analogs of these bases, including but notlimited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

[0060] As used herein, the term “oligonucleotide,” refers to a shortlength of single-stranded polynucleotide chain. Oligonucleotides aretypically less than 100 residues long (e.g., between 15 and 50),however, as used herein, the term is also intended to encompass longerpolynucleotide chains. Oligonucleotides are often referred to by theirlength. For example a 24 residue oligonucleotide is referred to as a“24-mer.” Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

[0061] As used herein, “recombinant nucleic acid,” “recombinant gene” or“recombinant DNA molecule” indicate that the nucleotide sequence orarrangement of its parts is not a native configuration, and has beenmanipulated by molecular biological techniques. The term implies thatthe DNA molecule is comprised of segments of DNA that have beenartificially joined together. Protocols and reagents to manipulatenucleic acids are common and routine in the art (See e.g., Maniatis etal.(eds.), Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY, [1982]; Sambrook et al. (eds.), Molecular Cloning:A Laboratory Manual, Second Edition, Volumes 1-3, Cold Spring HarborLaboratory Press, NY, [1989]; and Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, Vol. 1-4, John Wiley & Sons, Inc., NewYork [1994]).

[0062] Similarly, a “recombinant protein” or “recombinant polypeptide”refers to a protein molecule that is expressed from a recombinant DNAmolecule. Use of these terms indicates that the primary amino acidsequence, arrangement of its domains or nucleic acid elements whichcontrol its expression are not native, and have been manipulated bymolecular biology techniques. As indicated above, techniques tomanipulate recombinant proteins are also common and routine in the art.

[0063] The terms “exogenous” and “heterologous” are sometimes usedinterchangeably with “recombinant.” An “exogenous nucleic acid,”“exogenous gene” and “exogenous protein” indicate a nucleic acid, geneor protein, respectively, that has come from a source other than itsnative source, and has been artificially supplied to the biologicalsystem. In contrast, the terms “endogenous protein,” “native protein,”“endogenous gene,” and “native gene” refer to a protein or gene that isnative to the biological system, species or chromosome under study. A“native” or “endogenous” polypeptide does not contain amino acidresidues encoded by recombinant vector sequences; that is, the nativeprotein contains only those amino acids found in the polypeptide orprotein as it occurs in nature. A “native” polypeptide may be producedby recombinant means or may be isolated from a naturally occurringsource. Similarly, a “native” or “endogenous” gene is a gene that doesnot contain nucleic acid elements encoded by sources other than thechromosome on which it is normally found in nature.

[0064] As used herein, the term “portion” when in reference to a protein(as in “a portion of a given protein”) refers to fragments of thatprotein. The fragments may range in size from four amino acid residuesto the entire amino acid sequence minus one amino acid.

[0065] Nucleic acid molecules (e.g., DNA or RNA) are said to have “5′ends” and “3′ ends” because mononucleotides are reacted to makeoligonucleotides or polynucleotides in a manner such that the 5′phosphate of one mononucleotide pentose ring is attached to the 3′oxygen of its neighbor in one direction via a phosphodiester linkage.Therefore, an end of an oligonucleotides or polynucleotide, referred toas the “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide or polynucleotide, also may be said to have 5′ and 3′ends. In either a linear or circular DNA molecule, discrete elements arereferred to as being “upstream” or 5′ of the “downstream” or 3′elements. This terminology reflects the fact that transcription proceedsin a 5′ to 3′ fashion along the DNA strand. The promoter and enhancerelements that direct transcription of a linked gene are generallylocated 5′ or upstream of the coding region. However, enhancer elementscan exert their effect even when located 3′ of the promoter element orthe coding region. Transcription termination and polyadenylation signalsare located 3′ or downstream of the coding region.

[0066] The term “gene” refers to a nucleic acid (e.g., DNA) sequencecomprised of parts, that when appropriately combined in either a nativeor recombinant manner, provide some product or function. Genes may ormay not comprise coding sequences necessary for the production of apolypeptide. Examples of genes which do not encode polypeptide sequencesinclude ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. Genescan encode a polypeptide or any portion of a polypeptide within thegene's “coding region” or “open reading frame.” The polypeptide producedby the open reading frame of a gene may or may not display functionalactivity or properties of the full-length polypeptide (e.g., enzymaticactivity, ligand binding, signal transduction, etc.).

[0067] In addition to the coding region of the nucleic acid, the term“gene” also encompasses the transcribed nucleotide sequences of thefull-length mRNA adjacent to the 5′ and 3′ ends of the coding region.These noncoding regions are variable in size, and typically extend fordistances up to or exceeding 1 kb on both the 5′ and 3′ ends of thecoding region. The sequences that are located 5′ and 3′ of the codingregion and are contained on the mRNA are referred to as 5′ and 3′untranslated sequences (5′ UT and 3′ UT). Both the 5′ and 3′ UT mayserve regulatory roles, including translation initiation,post-transcriptional cleavage and polyadenylation. The term “gene”encompasses mRNA, cDNA and genomic forms of a gene.

[0068] It is contemplated that the genomic form or genomic clone of agene may contain the sequences of the transcribed mRNA, as well as othernon-coding sequences which lie outside of the mRNA. The regulatoryregions which lie outside the mRNA transcription unit are sometimescalled “5′ or 3′ flanking sequences.” A functional genomic form of agene must contain regulatory elements necessary for the regulation oftranscription. The term “promoter/enhancer region” is usually used todescribe this DNA region, typically but not necessarily 5′ of the siteof transcription initiation, sufficient to confer appropriatetranscriptional regulation. The word “promoter” alone is sometimes usedsynonymously with “promoter/enhancer.” A promoter may be constitutivelyactive, or alternatively, conditionally active, where transcription isinitiated only under certain physiological conditions or in the presenceof certain drugs. The 3′ flanking region may contain additionalsequences which regulate transcription, especially the termination oftranscription. “Introns” or “intervening regions” or “interveningsequences” are segments of a gene which are contained in the primarytranscript (i.e., hetero-nuclear RNA, or hnRNA), but are spliced out toyield the processed mRNA form. Introns may contain transcriptionalregulatory elements such as enhancers. The mRNA produced from thegenomic copy of a gene is translated in the presence of ribosomes toyield the primary amino acid sequence of the polypeptide.

[0069] As used herein, the term “regulatory element” refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element that enablesthe initiation of transcription of an operably linked coding region.Other regulatory elements are splicing signals, polyadenylation signals,termination signals, etc.

[0070] Transcriptional control signals in eukaryotes comprise “promoter”and “enhancer” elements. Promoters and enhancers consist of short arraysof DNA sequences that interact specifically with cellular proteinsinvolved in transcription (Maniatis et al., Science 236:1237 [1987]).Promoter and enhancer elements have been isolated from a variety ofeukaryotic sources including genes in yeast, insect and mammalian cells,as well as viruses. Analogous control elements (i.e., promoters andenhancers) are also found in prokaryotes. The selection of a particularpromoter and enhancer to be operably linked in a recombinant genedepends on what cell type is to be used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional only in a limited subset of cell types(for review see, Voss et al., Trends Biochem. Sci., 11:287 [1986] andManiatis et al., Science 236:1237 [1987]). For example, the SV40 earlygene enhancer is very active in a wide variety of mammalian cell types(Dijkema et al., EMBO J., 4:761 [1985]). Two other examples ofpromoter/enhancer elements active in a broad range of mammalian celltypes are those from the human elongation factor 1α gene (Uetsuki etal., J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene 91:217 [1990];Mizushima and Nagata, Nuc. Acids. Res., 18:5322 [1990]), the longterminal repeats of the Rous sarcoma virus (Gorman et al., Proc. Natl.Acad. Sci. USA 79:6777 [1982]), and human cytomegalovirus (Boshart etal., Cell 41:521 [1985]). Some promoter elements serve to direct geneexpression in a tissue-specific manner.

[0071] As used herein, the term “promoter/enhancer” denotes a segment ofDNA which contains sequences capable of providing both promoter andenhancer functions (i.e., the functions provided by a promoter elementand an enhancer element). For example, the long terminal repeats ofretroviruses contain both promoter and enhancer functions. Thepromoter/enhancer may be “endogenous,” or “exogenous,” or“heterologous.” An “endogenous” promoter/enhancer is one which isnaturally linked with a given gene in the genome. An “exogenous” or“heterologous” promoter/enhancer is one placed in juxtaposition to agene by means of genetic manipulation (i.e., molecular biologicaltechniques such as cloning and recombination) such that transcription ofthe gene is controlled by the linked promoter/enhancer.

[0072] The presence of “splicing signals” on an expression vector oftenresults in higher levels of expression of the recombinant transcript.Splicing signals mediate the removal of introns from the primary RNAtranscript and consist of a splice donor and acceptor site (See e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, New York [1989], pp. 16.7-16.8). Acommonly used splice donor and acceptor site is the splice junction fromthe 16S RNA of SV40.

[0073] Efficient expression of recombinant DNA sequences in eukaryoticcells requires the presence of signals directing the efficienttermination and polyadenylation of the resulting transcript.Transcription termination signals are generally found downstream of thepolyadenylation signal and are a few hundred nucleotides in length. Theterm “poly A site” or “poly A sequence” as used herein denotes a nucleicacid sequence that directs both the termination and polyadenylation ofthe nascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable as transcripts lacking a poly A tail areunstable and are rapidly degraded. The poly A signal utilized in anexpression vector may be “heterologous” or “endogenous.” An endogenouspoly A signal is one that is found naturally at the 3′ end of the codingregion of a given gene in the genome. A heterologous poly A signal isone that is isolated from one gene and placed 3′ of another gene. Acommonly used heterologous poly A signal is the SV40 poly A signal. TheSV40 poly A signal is contained on a 237 bp BamHI/BclI restrictionfragment and directs both termination and polyadenylation (See e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, New York [1989], pp.16.6-16.7).

[0074] The terms “in operable combination,” “in operable order,”“operably linked” and similar phrases when used in reference to nucleicacid herein are used to refer to the linkage of nucleic acid sequencesin such a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

[0075] As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene,” “polynucleotide having a nucleotide sequenceencoding a gene,” and similar phrases are meant to indicate a nucleicacid sequence comprising the coding region of a gene (i.e., the nucleicacid sequence which encodes a gene product). The coding region may bepresent in either a cDNA, genomic DNA or RNA form. When present in a DNAform, the oligonucleotide, polynucleotide or nucleic acid may besingle-stranded (i.e., the sense strand or the antisense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

[0076] As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” and similar phrases refer to theorder or sequence of deoxyribonucleotides along a strand ofdeoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the polypeptide (e.g.,protein) chain. The DNA sequence thus codes for the amino acid sequence.

[0077] As used herein, the term “gene expression” refers to the processof converting genetic information encoded in a gene into RNA (e.g.,mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e.,via the enzymatic action of an RNA polymerase), and for protein encodinggenes, into protein through “translation” of the mRNA. Gene expressioncan be regulated at many stages. “Up-regulation” or “activation” refersto regulation that increases the production of gene expression products(i.e., RNA or protein), while “down-regulation” or “repression” refersto regulation that decreases mRNA or protein production. Molecules(e.g., transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

[0078] As used herein, the terms “complementary” or “complementarity”are used in reference to polynucleotides (i.e., a sequence ofnucleotides) related by the rules of antiparallel base-pairing. Forexample, the sequence 5′-C-T-A-G-T-3′ is complementary to the sequence5′-A-C-T-A-G-3′. Complementarity may be “partial,” in which only some ofthe nucleic acids' bases are matched according to antiparallel basepairing rules. Also, there may be “complete” or “total” complementaritybetween two nucleic acids. The degree of complementarity between nucleicacid strands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in polymerase chain reaction (PCR) amplification reactions,as well as detection methods that depend upon binding between nucleicacids. As used herein, the terms “antiparallel complementarity” and“complementarity” are synonymous.

[0079] As used herein, the term “antisense” is used in reference to anynucleic acid which is antiparallel to and complementary to anothernucleic acid. Antisense DNA or RNA may be produced by any method. Forexample, a cDNA or a portion of a cDNA may be subcloned into anexpression vector containing a promoter which permits transcriptioneither in vitro or in vivo. The cDNA or a portion of the cDNA issubcloned in such a way that it is in the reverse orientation relativeto the direction of transcription of the cDNA in its native chromosome.Transcription of this antisense cDNA produces an RNA transcript that iscomplementary and antiparallel to the native mRNA. The mechanism bywhich an antisense nucleic acid produces effects in a biological systemis unclear, however, likely involves the formation of a duplex with itscomplementary nucleic acid within either the nucleus or cytoplasm of acell. These duplexes are theorized to block transcription of the nativemRNA or prevent its translation. Using antisense techniques, an“artificial knockout” mutant may be reproduced in an animal or animalcell line. The term “antisense strand” is used in reference to a nucleicacid strand that is complementary to the “sense” strand. The designation(−) (i.e., “negative”) is sometimes used in reference to the antisensestrand, with the designation (+) (i.e., “positive”) sometimes used inreference to the sense strand.

[0080] The following definitions are the commonly accepted definitionsof the terms “identity,” “similarity” and “homology.” Percent identityis a measure of strict amino acid conservation. Percent similarity is ameasure of amino acid conservation which incorporates both strictlyconserved amino acids, as well as “conservative” amino acidsubstitutions, where one amino acid is substituted for a different aminoacid having similar chemical properties (i.e. a “conservative”substitution). The term “homology” can pertain to either proteins ornucleic acids. Two proteins can be described as “homologous” or“non-homologous,” but the degree of amino acid conservation isquantitated by percent identity and percent similarity. Nucleic acidconservation is measured by the strict conservation of the basesadenine, thymine, guanine and cytosine in the primary nucleotidesequence. When describing nucleic acid conservation, conservation of thenucleic acid primary sequence is sometimes expressed as percenthomology. In the same nucleic acid, one region may show a highpercentage of nucleotide sequence conservation, while a different regioncan show no or poor conservation. Nucleotide sequence conservation cannot be inferred from an amino acid similarity score. Two proteins mayshow domains that in one region are homologous, while other regions ofthe same protein are clearly non-homologous.

[0081] As used herein, the term “hybridization” is used in reference tothe pairing of complementary nucleic acids. Hybridization can bedemonstrated using a variety of hybridization assays (Southern blot,Northern Blot, slot blot, phage plaque hybridization, and othertechniques). These protocols are common in the art (See e.g., Sambrooket al. (eds.), Molecular Cloning: A Laboratory Manual, Second Edition,Volumes 1-3, Cold Spring Harbor Laboratory Press, NY, [1989]; Ausubel etal. (eds.), Current Protocols in Molecular Biology, Vol. 1-4, John Wiley& Sons, Inc., New York [1994]). Hybridization is the process of onenucleic acid pairing with an antiparallel counterpart which may or maynot have 100% complementarity. Two nucleic acids which contain 100%antiparallel complementarity will show strong hybridization. Twoantiparallel nucleic acids which contain no antiparallel complementarity(generally considered to be less than 30%) will not hybridize. Twonucleic acids which contain between 31-99% complementarity will show anintermediate level of hybridization. A single molecule that containspairing of complementary nucleic acids within its structure is said tobe “self-hybridized.”

[0082] As used herein, the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acids hybridize.“Low or weak stringency” conditions are reaction conditions which favorthe complementary base pairing and annealing of two nucleic acids. “Highstringency” conditions are those conditions which are less optimal forcomplementary base pairing and annealing. The art knows well thatnumerous variables affect the strength of hybridization, including thelength and nature of the probe and target (DNA, RNA, base composition,present in solution or immobilized, the degree of complementary betweenthe nucleic acids, the T_(m) of the formed hybrid, and the G:C ratiowithin the nucleic acids). Conditions may be manipulated to define lowor high stringency conditions: factors such as the concentration ofsalts and other components in the hybridization solution (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)as well as temperature of the hybridization and/or wash steps.Conditions of “low” or “high” stringency are specific for the particularhybridization technique used.

[0083] During hybridization of two nucleic acids under high stringencyconditions, complementary base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences. Thus, conditions of “weak” or “low” stringency are oftenrequired with nucleic acids that are derived from organisms that aregenetically diverse, as the frequency of complementary sequences isusually less. As used herein, two nucleic acids which are able tohybridize under high stringency conditions are considered “substantiallyhomologous.”

[0084] Whether sequences are “substantially homologous” may be verifiedusing hybridization competition assays. For example, a “substantiallyhomologous” nucleotide sequence is one that at least partially inhibitsa completely complementary probe sequence from hybridizing to a targetnucleic acid under conditions of low stringency. This is not to say thatconditions of low stringency are such that non-specific binding ispermitted; low stringency conditions require that the binding of twosequences to one another be a specific (i.e., selective) interaction.The absence of non-specific binding may be verified by the use of asecond target that lacks even a partial degree of complementarity (e.g.,less than about 30% identity); in the absence of non-specific bindingthe probe will not hybridize to the second non-complementary target.When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of highstringency.

[0085] A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene contain regions of nucleotide sequenceidentity (100% homology), representing the presence of the same exon orportion of the same exon on both cDNAs, as well as regions of completenon-identity. Because the two cDNAs contain regions of sequence identitythey will both hybridize to a probe derived from the entire gene orportions of the gene containing sequences found on both cDNAs. As usedherein, the two splice variants are therefore substantially homologousto such a probe and to each other.

[0086] As used herein, the term “T_(m)” is used in reference to the“melting temperature.” The melting temperature is the temperature atwhich a population of double-stranded nucleic acid molecules becomeshalf dissociated into single strands. The equation for calculating theT_(m) of nucleic acids is well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (See e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization[1985]). Other references include more sophisticated computations thattake structural as well as sequence characteristics into account for thecalculation of T_(m).

[0087] As used herein, the term “amplifiable nucleic acid” is used inreference to nucleic acids which may be amplified by any amplificationmethod. It is contemplated that “amplifiable nucleic acid” will usuallycomprise “template.” As used herein, the term “template” refers tonucleic acid originating from a sample that is to be used as a substratefor the generation of the amplified nucleic acid.

[0088] As used herein, the term “primer” refers to an oligonucleotide,typically but not necessarily produced synthetically, that is capable ofacting as a point of initiation of nucleic acid synthesis when placedunder conditions in which synthesis of a primer extension product thatis complementary to a nucleic acid strand is induced, (i.e., in thepresence of nucleotides, an inducing agent such as DNA polymerase, andat a suitable temperature and pH). The primer is preferably singlestranded for maximum efficiency in amplification, but may alternativelybe double stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and theuse of the method.

[0089] As used herein, the term “probe” refers to an oligonucleotide(i.e., a sequence of nucleotides), whether produced as a purifiedrestriction digest or produced by synthetic means, recombinantly or byamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that in preferred embodiments, anyprobe used in the present invention will be labelled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as immunohistochemicalassays), fluorescent, radioactive, and luminescent systems. It is notintended that the present invention be limited to any particulardetection system or label.

[0090] As used herein, the term “polymerase chain reaction” (“PCR”)refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202and 4,965,188, each of which is hereby incorporated by reference, whichdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a thermostable DNApolymerase. The two primers are complementary to their respectivestrands of the double stranded target sequence. To effect amplification,the mixture is denatured and the primers then annealed to theircomplementary sequences within the target molecule. Following annealing,the primers are extended with a polymerase so as to form a new pair ofcomplementary strands. The steps of denaturation, primer annealing andpolymerase extension can be repeated many times (i.e., denaturation,annealing and extension constitute one “cycle”; there can be numerous“cycles”) to obtain a high concentration of an amplified segment of thedesired target sequence. The length of the amplified segment of thedesired target sequence is determined by the relative positions of theprimers with respect to each other, and therefore, this length is acontrollable parameter. By virtue of the repeating aspect of theprocess, the method is referred to as the “polymerase chain reaction”(hereinafter “PCR”). Because the desired amplified segments of thetarget sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be “PCR amplified”.

[0091] With PCR, it is possible to amplify a single copy of a specifictarget sequence in genomic DNA to a level detectable by severaldifferent methodologies (e.g., hybridization with a labelled probe;incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of ³²P-labelled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide or polynucleotide sequencecan be amplified with the appropriate set of primer molecules. Inparticular, the amplified segments created by the PCR process are,themselves, efficient templates for subsequent PCR amplifications.

[0092] As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

[0093] As used herein, the term “amplification reagents” refers to thosereagents (e.g., deoxyribonucleotide triphosphates, buffer, etc.), neededfor amplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

[0094] As used herein, the terms “restriction endonucleases” and“restriction enzymes” refer to bacterial enzymes, each of which cutdouble-stranded DNA at or near a specific nucleotide sequence.

[0095] The term “isolated” when used in relation to a nucleic acid, asin “an isolated nucleic acid,” “an isolated oligonucleotide” or“isolated polynucleotide” refers to a nucleic acid sequence that isidentified and separated from at least one contaminant nucleic acid withwhich it is ordinarily associated in its natural source. Isolatednucleic acid is present in a form or setting that is different from theform or setting of that nucleic acid found in nature. In contrast,non-isolated nucleic acids are found in the state in which they exist innature. For example, a given DNA sequence (e.g., a gene) is found on thehost cell chromosome in proximity to neighboring genes; RNA sequences,such as a specific mRNA sequence encoding a specific protein, are foundin the cell in a mixture with numerous other mRNAs that encode amultitude of proteins. However, isolated nucleic acid encoding a givenpolypeptide includes, by way of example, such nucleic acid in cellsordinarily expressing the given protein where the nucleic acid is in achromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid, oligonucleotide, or polynucleotidemay be present in single-stranded or double-stranded form. When anisolated nucleic acid, oligonucleotide or polynucleotide is to beutilized to express a protein, the oligonucleotide or polynucleotidewill contain at a minimum the sense or coding strand (i.e., theoligonucleotide or polynucleotide may be single-stranded), but maycontain both the sense and anti-sense strands (i.e., the oligonucleotideor polynucleotide may be double-stranded).

[0096] As used herein, the term “purified” or “to purify” refers to theremoval of contaminants from a sample. For example, antibodies arepurified by removal of contaminating non-immunoglobulin proteins; theyare also purified by the removal of immunoglobulin that does not bind tothe target molecule. The removal of non-immnunoglobulin proteins and/orthe removal of immunoglobulins that do not bind to the target moleculeresults in an increase in the percent of target-reactiveinmmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

[0097] As used herein, the term “vector” is used in reference to nucleicacid molecules that transfer DNA segment(s) from one cell to another.The term “vehicle” is sometimes used interchangeably with “vector.” Avector “backbone” comprises those parts of the vector which mediate itsmaintenance and enable its intended use (e.g., the vector backbone maycontain sequences necessary for replication, genes imparting drug orantibiotic resistance, a multiple cloning site, and possibly operablylinked promoter/enhancer elements which enable the expression of acloned nucleic acid). The cloned nucleic acid (e.g., such as a cDNAcoding sequence, or an amplified PCR product) is inserted into thevector backbone using common molecular biology techniques. Vectors areoften derived from plasmids, bacteriophages, or plant or animal viruses.A “cloning vector” or “shuttle vector” or “subcloning vector” containoperably linked parts which facilitate subcloning steps (e.g., amultiple cloning site containing multiple restriction endonucleasesites). A “recombinant vector” indicates that the nucleotide sequence orarrangement of its parts is not a native configuration, and has beenmanipulated by molecular biological techniques. The term implies thatthe vector is comprised of segments of DNA that have been artificiallyjoined.

[0098] The term “expression vector” as used herein refers to arecombinant DNA molecule containing a desired coding sequence andoperably linked nucleic acid sequences necessary for the expression ofthe operably linked coding sequence in a particular host organism (e.g.,a bacterial expression vector, a yeast expression vector or a mammalianexpression vector). Nucleic acid sequences necessary for expression inprokaryotes typically include a promoter, an operator (optional), and aribosome binding site, often along with other sequences. Eukaryoticcells utilize promoters, enhancers, and termination and polyadenylationsignals and other sequences which are different from those used byprokaryotes.

[0099] Eukaryotic expression vectors may also contain “viral replicons”or “viral origins of replication.” Viral replicons are viral DNAsequences that allow for the extrachromosomal replication of a vector ina host cell expressing the appropriate replication factors. Some vectorsreplicate their nucleic acid to high copy numbers (e.g., vectors thatcontain either the SV40 or polyoma virus origin of replication replicateto high “copy number” (up to 10⁴ copies/cell) in cells that express theappropriate viral T antigen). Other vectors replicate their nucleic acidin low copy numbers (e.g., vectors that contain the replicons frombovine papillomavirus or Epstein-Barr virus replicate extrachromosomallyat “low copy number” (˜100 copies/cell)). The viral origins ofreplication listed above are not limiting, as the art is aware of otherorigins of replication that are commonly used in eukaryotic expressionvectors.

[0100] The term “transgene” as used herein refers to a foreign gene thatis placed into an organism by, for example, introducing the foreign geneinto newly fertilized eggs or early embryos. The term “foreign gene”refers to any nucleic acid (e.g., gene sequence) that is introduced intothe genome of an animal by experimental manipulations and may includegene sequences found in that animal so long as the introduced gene doesnot reside in the same location as does the naturally-occurring gene.

[0101] Embryonal cells at various developmental stages can be used tointroduce transgenes for the production of transgenic, non-humananimals. Different methods are used depending on the stage ofdevelopment of the embryonic cell. The zygote is the best target formicro-injection. In the mouse, the male pronucleus reaches the size ofapproximately 20 micrometers in diameter which allows reproducibleinjection of 1-2 picoliters (pl) of DNA solution. The use of zygotes asa target for gene transfer has a major advantage in that in most casesthe injected DNA will be incorporated into the host genome before thefirst cleavage (Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442[1985]). As a consequence, all cells of the transgenic non-human animalwill carry the incorporated transgene. This will, in general, also bereflected in the efficient transmission of the transgene to offspring ofthe founder since 50% of the germ cells will harbor the transgene.Micro-injection of zygotes is the preferred method for incorporatingtransgenes in practicing the invention. U.S. Pat. No. 4,873,191, hereinincorporated by reference, describes a method for the micro-injection ofzygotes.

[0102] Retroviral infection can also be used to introduce transgenesinto a non-human animal. The developing embryo can be cultured in vitroto the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenisch, Proc. Natl. Acad. Sci. USA73:1260-1264 [1976]). Efficient infection of the blastomeres is obtainedby enzymatic treatment to remove the zona pellucida (See e.g., Hogan etal., in Manipulating the Mouse Embryo, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. [1986]). The viral vector system used tointroduce the transgene is typically a replication-defective retroviruscarrying the transgene (Jahner et al., Proc. Natl. Acad Sci. USA82:6927-693 [1985]). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (vander Putten, Proc. Natl. Acad. Sci. USA 82(18):6148-52 [1985]; andStewart, et al., EMBO J. 6:383-388 [1987]). Alternatively, infection canbe performed at a later stage. Virus or virus-producing cells can beinjected into the blastocoele (Jahner et al., Nature 298:623-628[1982]). Most of the founders will be mosaic for the transgene sinceincorporation occurs only in a subset of cells that form the transgenicanimal. Further, the founder may contain various retroviral insertionsof the transgene at different positions in the genome that generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germline, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (Jauner etal., Nature 298:623-628 [1982]). Additional means of using retrovirusesor retroviral vectors to create transgenic animals known to the artinclude, but are not limited to, the micro-injection of retroviralparticles or mitomycin C-treated cells producing retrovirus into theperivitelline space of fertilized eggs or early embryos (PCTInternational Application WO 90/08832 [1990], and Haskell and Bowen,Mol. Reprod. Dev., 40:386 [1995]).

[0103] A third type of target cell for transgene introduction is theembryonal stem (ES) cell. ES cells are obtained by culturingpre-implantation embryos in vitro under appropriate conditions (Evans etal., Nature 292:154-156 [1981]; Bradley et al., Nature 309:255-258[1984]; Gossler et al., Proc. Acad. Sci. USA 83:9065-9069 [1986]; andRobertson et al., Nature 322:445-448 [1986]). Transgenes can beefficiently introduced into the ES cells by DNA transfection using avariety of methods known to the art including calcium phosphateco-precipitation, protoplast or spheroplast fusion, lipofection andDEAE-dextran-mediated transfection. Transgenes may also be introducedinto ES cells by retrovirus-mediated transduction or by micro-injection.Such transfected ES cells can thereafter colonize an embryo followingtheir introduction into the blastocoel of a blastocyst-stage embryo andcontribute to the germ line of the resulting chimeric animal (forreview, see Jaenisch, Science 240:1468-1474 [1988]). Prior to theintroduction of transfected ES cells into the blastocoel, thetransfected ES cells may be subjected to various selection protocols toenrich for ES cells that have integrated the transgene, assuming thatthe transgene provides a means for such selection. Alternatively, PCRmay be used to screen for ES cells that have integrated the transgene.This technique obviates the need for growth of the transfected ES cellsunder appropriate selective conditions prior to transfer into theblastocoel.

[0104] The terms “overexpression” and “overexpressing” and grammaticalequivalents are used in reference to levels of mRNA or protein where thelevel of expression of the mRNA or protein is higher than that typicallyobserved in a given tissue in a control or non-transgenic animal. Levelsof mRNA or protein are measured using any of a number of techniquesknown to those skilled in the art. For example, mRNA levels may beassayed using (but not limited to) a Northern blot analysis. Appropriatecontrols are included on the Northern blot to control for differences inthe amount of RNA loaded from each tissue analyzed (e.g., the amount of28S rRNA, an abundant RNA transcript present at essentially the sameamount in all tissues, present in each sample can be used as a means ofnormalizing or standardizing the mRNA-specific signal observed onNorthern blots). The amount of mRNA present in the band corresponding insize to the correctly spliced transgene RNA is quantified; other minorspecies of RNA which hybridize to the transgene probe are not consideredin the quatntification of the expression of the transgenic mRNA.

[0105] The term “transfection” as used herein refers to the introductionof foreign DNA into cells. Transfection may be accomplished by a varietyof means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.Mammalian cell transfection-techniques are common in the art, and aredescribed in many sources (See, e.g., Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, Vol. 1-4, John Wiley & Son, Inc., NewYorK [1994]).

[0106] The term “stable transfection” or “stably transfected” refers tothe introduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell whichcontains stably integrated foreign DNA within its own genomic DNA.

[0107] The term “transient transfection” or “transiently transfected”refers to the introduction of foreign DNA into a cell where the foreignDNA fails to integrate into the genome of the transfected cell. Theforeign DNA persists in the nucleus of the transfected cell for severaldays. During this time the foreign DNA is subject to the regulatorycontrols that govern the expression of endogenous genes in thechromosomes. The term “transient transfectant” refers to cells whichhave taken up foreign DNA but have failed to integrate this DNA.

[0108] The term “calcium phosphate co-precipitation” refers to atechnique for the introduction of nucleic acids into a eukaryotic cell,and most typically mammalian cells. The uptake of nucleic acids by cellsis enhanced when the nucleic acid is presented as a calciumphosphate-nucleic acid co-precipitate. Various modifications of theoriginal technique of Graham and van der Eb (Graham and van der Eb,Virol., 52:456 [1973]) are known in which the conditions for thetransfection of a particular cell type has been optimized. The art iswell aware of these various methods.

[0109] The term “transformation” has multiple meanings, depending on itsusage. In one sense, the term “transformation” is used to describe theprocess of introduction of foreign DNA into prokaryotic cells (i.e.,bacterial cells), and most frequently E. coli strains. Bacterial celltransformation may be accomplished by a variety of means well known tothe art, including the preparation of “competent” bacteria by the use ofcalcium chloride, magnesium chloride or rubidium chloride, andelectroporation. When a plasmid is used as the transformation vector,the plasmid typically contains a gene conferring drug resistance, suchas the genes encoding ampicillin, tetracycline or kanamycin resistance.Bacterial transformation techniques are common in the art, and aredescribed in many sources (e.g., Cohen et al., Proc. Natl. Acad. Sci.USA 69: 2110-2114 [1972]; Hanahan, J. Mol. Biol., 166:557-580 [1983];Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, SecondEdition, Volumes 1-3, Cold Spring Harbor Laboratory Press, NY, [1989];Ausubel et al. (eds.), Current Protocols in Molecular Biology, Vol. 1-4,John Wiley & Sons, Inc., New York [1994]).

[0110] “Transformation” also describes the physiological process bywhich a normal eukaryotic cell acquires the phenotypic qualities of amalignant cell. Such properties can include the ability to grow in softagar, the ability to grow in nutrient poor conditions, rapidproliferation, and the loss of contact inhibition. A eukaryotic cellwhich is “transformed” displays the properties of malignant cells. Aeukaryotic cell may acquire its transformed phenotype in vivo, or beartificially transformed in culture.

[0111] As used herein, the term “selectable marker” refers to the use ofa gene that encodes an enzymatic activity that confers the ability togrow in medium lacking what would otherwise be an essential nutrient(e.g., the HIS3 gene in yeast cells); in addition, a selectable markermay confer resistance to an antibiotic or drug upon the cell in whichthe selectable marker is expressed. Furthermore, selectable markers maybe “dominant.” Dominant selectable markers encode an enzymatic activitythat can be detected in any eukaryotic cell line. Examples of dominantselectable markers include the bacterial aminoglycoside 3′phosphotransferase gene (i.e., the neo gene) that confers resistance tothe drug G-418 in mammalian cells, as well as the bacterial hygromycin Gphosphotransferase (hyg) gene that confers resistance to the antibiotichygromycin, and the bacterial xanthine-guanine phosphoribosyltransferase gene (i.e., the gpt gene) that confers the ability to growin the presence of mycophenolic acid. The use of non-dominant selectablemarkers must be in conjunction with a cell line that lacks the relevantenzyme activity. Examples of non-dominant selectable markers include thethymidine kinase (tk) gene (used in conjunction with tk⁻ cell lines),the CAD gene (used in conjunction with CAD-deficient cells) and themammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) gene(used in conjunction with hprt⁻ cell lines). A review of the use ofselectable markers in mammalian cell lines is provided in Sambrook etal., Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, New York (1989), at pp.16.9-16.15.

[0112] As used herein in the context of protein purification, the terms“source culture,” “starting culture,” “starting material” or “culture”or the like can include any of the following materials: culturesupernatant, cultured eukaryotic or prokaryotic cells (e.g., animalcells or bacteria), crushed eukaryotic or prokaryotic cells, tissueremoved from an organism, or the product of an in vitro translation orin vitro coupled transcription/translation reaction. The cells of such aculture may or may not contain recombinant nucleic acid.

[0113] As used herein, the term “cell culture” refers to any in vitroculture of cells. Included within this term are continuous cell lines(e.g., with an immortal phenotype), primary cell cultures, finite celllines (e.g., non-transformed cells), and any other cell populationmaintained in vitro.

[0114] The term “test compound” refers to any chemical entity,pharmaceutical, drug, composition, and the like that can be used totreat or prevent a disease, illness, sickness, or disorder of bodilyfunction. Test compounds comprise both known and potential therapeuticcompounds. A “known therapeutic compound” refers to a therapeuticcompound that has been previously described. Particularly preferredknown therapeutics are those that have been shown (e.g., through animaltrials or prior experience with administration to humans) to beeffective in treatment or prevention of pathology.

[0115] As used herein, a “drug” can be any molecule of any composition,including protein, peptide, nucleic acid, organic molecule, inorganicmolecule, or combinations of molecules, biological or non-biological,which are capable of producing a physiological response. As used herein,a “drug” provides at least one beneficial response in the cure,mitigation, treatment or prevention of a disease or disorder. A compoundis considered a “drug candidate” if it is not yet known if that compoundwill provide at least one beneficial response in the cure, mitigation,treatment or prevention of a disease, disorder or condition. A “druglibrary” is a collection of molecules, where it may or may not be knownif one or multiple drugs in the library have therapeutic value.

[0116] As used herein, the terms “host,” “expression host,” and“transformant” refer to organisms and/or cells which harbor an exogenousDNA sequence (e.g., via transfection), an expression vector or vehicle,as well as organisms and/or cells that are suitable for use inexpressing a recombinant gene or protein. It is not intended that thepresent invention be limited to any particular type of cell or organism.Indeed, it is contemplated that any suitable organism and/or cell willfind use in the present invention as a host.

[0117] As used herein, the term “subject” refers to any animal beingexamined, studied or treated. It is not intended that the presentinvention be limited to any particular type of subject. It iscontemplated that multiple organisms will find use in the presentinvention as subjects. In some embodiments, humans are the preferredsubject.

[0118] A subject displaying pathology resulting from abnormal cytokineactivity may display symptoms that include, but are not limited to,inflammation, cachexia, insulin resistance, overstimulation ofinterleukin-6 and granulocyte/macrophage-colony stimulating factor(GM-CSF) secretion, enhanced cytotoxicity of polymorphonuclearneutrophils, prolonged expression of cellular adhesion molecules,induction of procoagulant activity on vascular endothelial cells,increased adherence of neutrophils and lymphocytes, stimulation of therelease of platelet activating factor from macrophages, neutrophils andvascular endothelial cells, fever, malaise, and anorexia.

[0119] As used herein, a “disease” is a disruption of normal bodyfunction, generally where two of three criteria are met: 1) theetiological agent is known, 2) an identifiable group of symptomsappears, and 3) there are consistent anatomical or physiologicalalterations. Examples include, but are not limited to rheumatoidarthritis, inflammatory bowel disease and graft-versus-host disease. Adisorder is, in general, a disruption of some aspect of normal bodyfunction (e.g., rheumatoid arthritis and inflammatory bowel disease areimmune disorders).

[0120] As used herein, the term “antigen” refers to any agent (e.g., anysubstance, compound, molecule [including macromolecules], or othermoiety), that is recognized by an antibody, while the term “immunogen”refers to any agent (e.g., any substance, compound, molecule [includingmacromolecules], or other moiety) that can elicit an immunologicalresponse in an individual. These terms may be used to refer to anindividual macromolecule or to a homogeneous or heterogeneous populationof antigenic macromolecules. It is intended that the terms antigen andimmunogen encompass protein molecules or at least one portion of aprotein molecule, which contains one or more epitopes. In many cases,antigens are also immunogens, thus the term “antigen” is often usedinterchangeably with the term “immunogen.” The substance may then beused as an antigen in an assay to detect the presence of appropriateantibodies in the serum of the immunized animal.

[0121] The term “antigenic determinant” as used herein refers to thatportion of an antigen that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

[0122] The terms “specific binding” or “specifically binding” when usedin reference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general.

[0123] As used herein, the term “adjuvant” is defined as a substanceknown to increase the immune response to other antigens whenadministered with other antigens. If adjuvant is used, it is notintended that the present invention be limited to any particular type ofadjuvant—or that the same adjuvant, once used, be used all the time. Itis contemplated that adjuvants may be used either separately or incombination. The present invention contemplates all types of adjuvant,including but not limited to agar beads, aluminum hydroxide or phosphate(alum), Incomplete Freund's adjuvant (incomplete or complete), as wellas Quil A adjuvant and Gerbu adjuvant (Accurate Chemical and ScientificCorporation), and bacterins (i.e., killed preparations of bacterialcells). It is further contemplated that the vaccine comprise at leastone “excipient” (i.e., a pharmaceutically acceptable carrier orsubstance) suitable for administration to a human or other animalsubject. It is intended that the term “excipient” encompass liquids, aswell as solids, and colloidal suspensions.

[0124] As used herein the term “immunogenically-effective amount” refersto that amount of an immunogen required to invoke the production ofprotective levels of antibodies in a host upon vaccination.

[0125] The term “protective level,” when used in reference to the levelof antibodies induced upon immunization of the host with an immunogenmeans a level of circulating antibodies sufficient to protect the hostfrom challenge with a lethal dose of an organism or other material(e.g., toxins, etc.).

[0126] The terms “self antigen” or “autoantigen” refer to an antigen ora molecule normally expressed by an individual, but which solicits animmune response. Under normal conditions, these autoantigens arerecognized during an immune response as self (i.e., an antigen that isnormally part of the individual), and do not solicit an immune response.This is in contrast to antigens which are foreign, or exogenous, and arethus not normally part of the individual's antigenic makeup. “Selfantigen” or “autoantigen” is recognized as foreign, although the antigenis native to the individual's physiology.

[0127] As used herein, the term “autoimmune disease” means a set ofsustained organ-specific or systemic clinical symptoms and signsassociated with altered immune homeostasis that is manifested byqualitative and/or quantitative defects of expressed autoimmunerepertoires. Autoimmune diseases are characterized by antibody orcytotoxic immune responses to epitopes on self antigens. The immunesystem of the individual then activates an inflammatory cascade aimed atcells and tissues presenting those specific self antigens. Thedestruction of the antigen, tissue, cell type, or organ attacked by theindividual's own immune system gives rise to the signs and symptoms ofthe disease. Clinically significant autoimmune diseases include, forexample, rheumatoid arthritis, multiple sclerosis, juvenile-onsetdiabetes, systemic lupus erythematosus (SLE), autoimmune uveoretinitis,autoimmune vasculitis, bullous pemphigus, myasthenia gravis, autoimmunethyroiditis or Hashimoto's disease, Sjogren's syndrome, granulomatousorchitis, autoimmune oophoritis, Crohn's disease, sarcoidosis, rheumaticcarditis, ankylosing spondylitis, glomerulonephritis, Grave's disease,and autoimmune thrombocytopenic purpura.

[0128] The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays,including but not limited to lymphoproliferation (ie., lymphocyteactivation) assays, CTL cytotoxic cell assays such as chromium-releaseassays, or by assaying for T lymphocytes specific for the antigen in asensitized subject. Such assays are well known in the art (See e.g.,Erickson et al., J. Immunol., 151:4189-4199 [1993] and Doe et al., Eur.J. Immunol., 24:2369-2376 [1994]).

[0129] The term “modulate,” as used herein, refers to a change in thebiological activity of a biologically active molecule. Modulation can bean increase or a decrease in activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties of biologically active molecules.

[0130] The term “agonist,” as used herein, refers to a molecule which,when interacting with an biologically active molecule, causes a change(e.g., enhancement) in the biologically active molecule, which modulatesthe activity of the biologically active molecule. Agonists may includeproteins, nucleic acids, carbohydrates, or any other molecules whichbind or interact with biologically active molecules. For example,agonists can alter the activity of gene transcription by interactingwith RNA polymerase directly or through a transcription factor.

[0131] The terms “antagonist” or “inhibitor,” as used herein, refer to amolecule which, when interacting with a biologically active molecule,blocks or modulates the biological activity of the biologically activemolecule. Antagonists and inhibitors may include proteins, nucleicacids, carbohydrates, or any other molecules that bind or interact withbiologically active molecules. Inhibitors and antagonists can effect thebiology of cells, tissues, organs or entire organisms.

[0132] The terms “Westem blot,” “Western immunoblot” “immunoblot” and“Western” refer to the immunological analysis of protein(s),polypeptides or peptides that have been immobilized onto a membranesupport. The proteins are first resolved by polyacrylamide gelelectrophoresis (i.e., SDS-PAGE) to separate the proteins, followed bytransfer of the protein from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized proteins are thenexposed to an antibody having reactivity towards an antigen of interest.In preferred embodiments, the binding of the antibody (i.e., the primaryantibody) is detected by use of a secondary antibody which specificallybinds the primary antibody. The secondary antibody is typicallyconjugated to an enzyme which permits visualization of theantigen-antibody complex by the production of a colored reaction productor catalyzes a luminescent enzymatic reaction (e.g., the ECL reagent,Amersham).

[0133] As used herein, the term “ELISA” refers to enzyme-linkedimmunosorbent assay (or EIA). Numerous ELISA methods and applicationsare known in the art, and are described in many references (See e.g.,Crowther, “Enzyme-Linked Immunosorbent Assay (ELISA),” in MolecularBiomethods Handbook, Rapley et al. [eds.], pp. 595-617, Humana Press,Inc., Totowa, N.J. [1998]; See also, Harlow and Lane (eds.), Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press [1988]; andAusubel et al. (eds.), Current Protocols in Molecular Biology, Ch. 11,John Wiley & Sons, Inc., New York [1994], for general descriptions ofELISA methodology). In addition, there are numerous commerciallyavailable ELISA test systems, equipment and individual reagents.

[0134] One ELISA method is a “direct ELISA,” where an antigen (e.g.,sTNFR1 or ARTS-1) in a sample is detected. In one embodiment of thedirect ELISA, a sample containing antigen is exposed to a solid (i.e.,stationary or immobilized) support (e.g., a microtiter plate well). Theantigen within the sample becomes immobilized to the stationary phase,and is detected directly using an enzyme-conjugated antibody specificfor the antigen.

[0135] In an alternative embodiment, an antibody specific for an antigenis detected in a sample. In this embodiment, a sample containing anantibody (e.g., anti-ARTS-1 antiserum) is immobilized to a solid support(e.g., a microtiter plate well). The antigen-specific antibody issubsequently detected using purified antigen and an enzyme-conjugatedantibody specific for the antigen.

[0136] In further alternative embodiments, an “indirect ELISA” is usedto detect antibody or antigen in samples. In one embodiment, an antigen(or antibody) is immobilized to a solid support (e.g., a microtiterplate well) as in the direct ELISA, but is detected indirectly by firstadding an antigen-specific antibody (or antigen), then followed by theaddition of a detection antibody specific for the antibody thatspecifically binds the antigen, also known as “species-specific”antibodies (e.g., a goat anti-rabbit antibody), which are available fromvarious manufacturers known to those in the art (e.g., Santa CruzBiotechnology; Zymed and Pharmingen/Transduction Laboratories).

[0137] In other embodiments, a “sandwich ELISA” is used, where theantigen is immobilized on a solid support (e.g., a microtiter plate) viaan antibody (i.e., a capture antibody) that is immobilized on the solidsupport and is able to bind the antigen of interest. Following theaffixing of a suitable capture antibody to the immobilized phase, asample is then added to the microtiter plate well, followed by washing.If the antigen of interest is present in the sample, it is bound to thecapture antibody present on the support. In some embodiments, a sandwichELISA is a “direct sandwich” ELISA, where the captured antigen isdetected directly by using an enzyme-conjugated antibody directedagainst the antigen. Alternatively, in other embodiments, a sandwichELISA is an “indirect sandwich” ELISA, where the captured antigen isdetected indirectly by using an antibody directed against the antigen,which is then detected by another enzyme-conjugated antibody which bindsthe antigen-specific antibody, thus forming anantibody-antigen-antibody-antibody complex. Suitable reporter reagentsare then added to detect the third antibody. Alternatively, in someembodiments, any number of additional antibodies are added as necessary,in order to detect the antigen-antibody complex. In some preferredembodiments, these additional antibodies are labelled or tagged, so asto permit their visualization and/or quantitation.

[0138] As used herein, the term “capture antibody” refers to an antibodythat is used in a sandwich ELISA to bind (i.e., capture) an antigen in asample prior to detection of the antigen. In one embodiment of thepresent invention, biotinylated capture antibodies are used in thepresent invention in conjunction with avidin-coated solid support.Another antibody (i.e., the detection antibody) is then used to bind anddetect the antigen-antibody complex, in effect forming a “sandwich”comprised of antibody-antigen-antibody (i.e., a sandwich ELISA).

[0139] As used herein, a “detection antibody” is an antibody whichcarries a means for visualization or quantitation. Typically, detectionantibodies are conjugated enzyme moieties that yield a colored,fluorescent, or luminescent reaction product following the addition of asuitable substrate. Conjugated enzymes commonly used with detectionantibodies in the ELISA include, but are not limited to horseradishperoxidase, urease, alkaline phosphatase, glucoamylase andβ-galactosidase. In some embodiments, the detection antibody is directedagainst the antigen of interest, while in other embodiments, thedetection antibody is not directed against the antigen of interest.Thus, in some embodiments, the detection antibody is directed againstanother antibody. In some embodiments, the detection antibody is ananti-species antibody. Alternatively, the detection antibody is preparedwith a label (e.g., biotin, a fluorescent marker, or a radioisotope),and is detected and/or quantitated using this label.

[0140] As used herein, the terms “reporter reagent,” “reportermolecule,” “detection substrate” and “detection reagent” are used inreference to reagents which permit the detection and/or quantitation ofan antibody bound to an antigen. For example, in some embodiments, thereporter reagent is a colorimetric substrate for an enzyme that has beenconjugated to an antibody. Addition of a suitable substrate to theantibody-enzyme conjugate results in the production of a colorimetric,flubrimetric, or luminescent signal (e.g., following the binding of theconjugated antibody to the antigen of interest). Other reporter reagentsinclude, but are not limited to, radioactive compounds. This definitionalso encompasses the use of biotin and avidin-based compounds (e.g.,including but not limited to neutravidin and streptavidin) as part ofthe detection system.

[0141] As used herein, the term “signal” is used generally in referenceto any detectable process that indicates that a reaction has occurred,for example, binding of antibody to antigen. It is contemplated thatsignals in the form of radioactivity, fluorimetric or colorimetricproducts/reagents will all find use with the present invention. Invarious embodiments of the present invention, the signal is assessedqualitatively, while in alternative embodiments, the signal is assessedquantitatively.

[0142] As used herein, the term “amplifier” is used in reference to asystem which enhances the signal in a detection method, such as an ELISA(e.g., an alkaline phosphatase amplifier system used in an ELISA).

[0143] As used herein, the term “solid support” is used in reference toany solid or stationary material to which reagents such as antibodies,antigens, and other test components are attached. For example, inpreferred ELISA methods, the wells of microtiter plates provide solidsupports. Other examples of solid supports include microscope slides,coverslips, beads, particles, cell culture flasks, as well as any othersuitable items.

[0144] As used herein, the term “kit” is used in reference to acombination of reagents and other materials which facilitate sampleanalysis. In various embodiments of the present invention, a kit caninclude antibodies (e.g., a suitable capture antibody, reporterantibody, primary antibody, secondary antibody, detection antibody)purified control antigen, detection reagents, amplifier system, andnucleic acid probes. Furthermore, in other embodiments, the kitincludes, but is not limited to, components such as apparatus for samplecollection, sample tubes, holders, trays, racks, dishes, plates,instructions on the use of the kit, solutions or other chemicalreagents, and samples to be used for standardization, normalization,and/or control samples. It is contemplated that kits of the presentinvention can also include apparatus and reagents for electrophoresisand blotting.

[0145] As used herein, the term “in vitro” refers to an artificialenvironment and to processes or reactions that occur within anartificial environment. In vitro environments consist of, but are notlimited to, controlled laboratory conditions. The term “in vivo” refersto the natural environment (e.g., an animal or a cell) and to processesor reactions that occur within that natural environment.

[0146] As used herein, the terms “local” or “localized” and the likerefer to confinement to a small area, a single tissue, a single organ(e.g., a lung) or other defined structure.

[0147] As used herein, the term “localized delivery” is delivery of anagent (e.g. a gene therapy agent or a drug) to a small area, a singletissue, a single organ or other specific structure. For example,localized delivery of a gene therapy agent to a single site (e.g., theliver) in a subject is typically achieved by injection into that site.

[0148] As used herein, the term “systemic” refers to multiple sites,tissues or organs in an organism, or to the entire organism. Use of theword “systemic” generally indicates involvement of the circulatory orlymphatic systems.

[0149] As used herein, the term “systemic delivery” (in contrast tolocalized delivery) is delivery of an agent (e.g., a drug) to multiplesites, tissues or organs in an organism, or to the entire organism viathe circulatory system following an intravenous injection, or viagastrointestinal absorption of an orally administered agent.

[0150] As used herein, the term “surgical delivery” refers to thedelivery of an agent (e.g., a gene therapy agent) by surgical means(i.e., by operation or some other invasive manipulation). Thus, in someembodiments, surgical techniques provide means for localized delivery ofan agent.

DESCRIPTION OF THE INVENTION

[0151] The present invention provides compositions and methods relatedto regulation of cytokine signaling through the Tumor Necrosis Factor(TNF) pathway. Specifically, the present invention provides a novelpolypeptide, and a gene which encodes the polypeptide, as set forth inFIG. 1 and SEQ ID NOS:1 and 2, which has the ability to promote theshedding of the extracellular domain of Type I Tumor Necrosis FactorReceptor (TNFR1). This polypeptide and gene are called ARTS-1, foraminopeptidase regulator of type I, 55 kDa tumor necrosis factorreceptor ectodomain shedding. It is contemplated that methods whichregulate the shedding of the sTNFR1 also regulate the activity of TNF.It is further contemplated that the ARTS-1 gene, as well as genessubstantially homologous to the ARTS-1 gene (and the gene product)regulate ectodomain shedding of other cytokine receptors, including IL-1and IL-6. It is also contemplated that the compositions and methodsprovided by the present invention will find use in therapeutics for thetreatment of diseases and disorders resulting from aberrant TNFactivity.

TNFR1 Shedding

[0152] The complete mechanisms underlying TNFR1 ectodomain shedding areunknown, but are thought to be mediated via proteolytic cleavage of thespacer region located between the transmembrane and TNF-ligand bindingdomain (Brakebusch et al., J. Biol. Chem., 269(51):32488-96 [1994]).Based upon amino acid sequencing of sTNFR1 isolated from urine, themajor cleavage site has been identified as occurring between Asn-180 andVal-181, with a minor site located between Lys-182 and Gly-183 (Nopharet al., EMBO J., 9:3269-3278 [1990]; Wallach et al., In Tumor NecrosisFactor: Structure-Function Relationship and Clinical Application (Osawaand Baonavida, eds.) Vol III,. 47-57, S. Karger, Basel, Switzerland[1991]; and Brakebusch et al., J. Biol. Chem., 269(51):32488-96 [1994]).However, an understanding of the mechanism(s) is not necessary in orderto use the present invention.

[0153] Again, although an understanding of the mechanism(s) is notnecessary in order to use the present invention, a completeunderstanding of the mechanism regulating TNFR1 shedding requires theidentification and characterization of the interactions betweenregulatory proteins, such as ARTS-1, TNFR1, and TNFR1 receptorsheddases, which appear to belong to the metalloprotease-disintegrin(ADAM) family of zinc metalloproteases (Blobel et al, Cell 90:589-592[1997]). For example, TNF-α converting enzyme (TACE, ADAM17) has beenreported to mediate the ectodomain shedding of TNF-α, tranforming growthfactor-α, L-selectin and TNFR2 (Black et al., Nature 385:729-733 [1997];Moss et al., Nature 385:733-736 [1997]; and Peschon et al., Science282:1281-1284 [1998]). Similarly, TACE has been implicated in theregulated α-secretase cleavage of the amyloid precursor protein andectodomain shedding of erbB4/HER4 (Buxbaum et al, J. Biol. Chem.,273:27765-27767 [1998]; Rio et al., J. Biol. Chem., 275:10379-10387[2000]). Recent data suggest that TACE may also mediate TNFR1 ectodomainshedding based upon the demonstration of increased TNFR1 shedding inreconstituted TACE-deficient cell lines (Reddy et al., J. Biol. Chem.,275:14608-14614 [2000]). Indeed, TACE has been implicated as havingsheddase activity toward both TNFR1 and IL-1R type II (Reddy et al.,supra).

[0154] Studies have also been undertaken to identify enzymes capable ofcleaving (i.e., shedding) the leucocyte selectin (L-selectin) receptor,a peripheral lymph node homing receptor. The enzymes which shed theL-selectin receptor have been theorized to be metalloproteases (Preeceet al., J. Biol. Chem., 271:11634-11640 [1996]; Peschon et al., Science282:1281-1284 [1998]; and Borland et al., J. Biol. Chem., 274:2810-2815[1999]).

[0155] Neither the sheddase nor the mechanism regulating the shedding ofsTNFR1 are known, although attempts have been made to identify sTNFR1shedding activities in crude preparations. However, the results indicatemetal requirements for these activities, thereby tentativelycategorizing the enzymes as metalloproteases (Bjonberg et al., Scand. J.Immunol., 42:418-424 [1995]; Mullberg et al., J. Immunol., 155:5198-5205[1995]; Katsura et al., Biochem. Biophys. Res. Comm., 222:298-302[1996]; Williams et al., J. Clin. Invest., 97(12):2833-2841 [1996]; andGallea-Robache et al., Cytokine 9:340-346 [1997]). However, themolecular identities of these metalloprotease enzymes remain unknown.

[0156] Björnberg et al. (Björnberg et al., Scand. J. Immunol.,42:418-424 [1995]) indicate that in assays to identify and characterizeenzymes involved in TNFR1 processing, inhibitors of aminopeptidases hadnegligible effects on release of sTNFR1. These authors indicate thataminopeptidases are not involved in release of sTNFR1.

[0157] The mechanisms which regulate TNF activity, TNFR activation,TNFR1 signaling, sTNFR1 activity and sTNFR1 shedding are poorlyunderstood. However, an understanding of these mechanisms is notnecessary to practice the present invention. Indeed, the presentinvention is not limited to any particular mechanism or mechanisms.

IL-1RII and IL-6R Shedding

[0158] As discussed above, the cognate receptors for the cytokines IL-1and IL-6 also exhibit soluble forms akin to the soluble form of TNFR1.Furthermore, it has been suggested that these soluble receptor formsplay a role in the regulation of IL-1 and IL-6 activity andpro-inflammatory response. However, the proteins responsible for theshedding of ectodomains of the receptors for IL-1 and IL-6 remainunidentified. Nonetheless, it is contemplated that a protein whichregulates the shedding of TNFR1 ectodomain (e.g., a protein of thepresent invention) will also regulate the shedding of ectodomains ofother cytokine receptors, including IL-1RII and IL-6R.

Anti-TNF Therapeutic Strategies

[0159] The recognition of TNF as an important inflammatory mediator inboth health and disease has fostered the development of a variety oftherapeutic strategies directed at inhibiting TNF bioactivity.

[0160] The benefits of inhibiting TNF activity during inflammatoryreactions have been demonstrated using neutralizing monoclonalantibodies to TNF (Tracey et al., Nature 330:662-664 [1987]; Hinshaw etal., Circ. Shock 30:279-292 [1990]; Opal et al., J. Infect. Dis.,161:1148-1152 [1990]; Silva et al., J. Infect. Sis., 162:421-427 [1990];Emerson et al., Circ. Shock 38:75-84 [1992]; Fieldler et al., J. Lab.Clin. Med., 120:574-588 [1992]; Jesmok et al., Am. J. Pathol.,141:1197-1207 [1992]; Walsh et al., Arch. Surg., 127:138-144 [1992]; andWilliams et al., Proc. Natl. Acad. Sci. USA 89:9784-9788 [1992]).Unfortunately, the development of widespread clinical application ofneutralizing monoclonal antibodies directed against human TNF ishampered by the potential for immune rejection of the mouse anti-TNFantibodies in human hosts. The development of an immune response tonon-human anti-TNF antibodies administered to human subjects maydecrease the duration of therapeutic efficacy and also result in adverseevents related to the formation of immune complexes or the developmentof hypersensitivity (Kempeni, Ann. Rheum. Dis., 58(S1):I73-I81 [1999]).

[0161] A chimeric monoclonal antibody consisting of the variable regionof a murine anti-TNF monoclonal antibody fused to the constant region ofIgG1k has also been studied in clinical trials for the treatment ofCrohn's disease (i.e., inflammatory bowel disease) (Knight et al., Mol.Immunol., 30(16):1443-53 [1993]; Targan et al., N. Engl. J. Med.,337:1029-1035 [1997]; and U.S. Pat. No. 5,656,272 to Le et al., herebyincorporated by reference). However, this anti-TNF antibody is notoptimal, due to the likely development of human anti-chimeric antibodiesdirected against the non-human elements or the artificially fusedsequences within the chimeric anti-TNF antibody. These deleteriousantibodies are likely to reduce the half-life and therapeutic efficacyof the chimeric antibody, and possibly result in the formation ofunwanted immune complexes or the development of hypersensitivity (Targanet al., N. Engl. J. Med., 337:1029-1035 [1997]; Harriman et al., Ann.Rheum. Dis., 58:161-164 [1999]; and Kempeni, J. Ann. Rheum. Dis.,58:I73-I81 [1999]).

[0162] Anti-TNF therapies utilizing TNF receptor fragments and chimeric,soluble fusion proteins consisting of TNF receptor and IgG Fc, have beenreported to be efficacious for patients with rheumatoid arthritis andinflammatory bowel disease (Moreland et al., New Engl. J. Med.,337:141-147 [1997]; and U.S. Pat. No. 5,605,690 to Jacobs et al. (herebyincorporated by reference)). However, the therapeutic efficacy of theseanti-TNF therapeutic strategies utilizing soluble forms of the TNFreceptor or chimeric antibodies are also likely to be limited by thedevelopment of an immune response to the artificially fused humansequences (Kempeni, Ann. Rheum. Dis., 58(S1):I73-I181 [1999]).

[0163] Current methods of controlling TNF activity in patients alsoinclude the use of non-specific inhibitors of cytokine genetranscription such as corticosteroids (e.g., dexamethasone andcyclosporin-A). These methods have significant and well known toxic sideeffects which result in significant discomfort and potentiallylife-threatening susceptibility to infection, as well as tissue andorgan damage. Toxicities associated with cyclosporin-A includenephrotoxicity, hypertension, hepatic toxicity, neurotoxicity,hirsutism, gingival hyperplasia and gastrointestinal toxicity (Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9^(th) edition,McGraw-Hill, NY, [1996], p. 1299). Toxicity associated withcorticosteroids include adrenal suppression, hyperglycemia,hypertension, edema, hypokalemic alkalosis, myopathy, peptic ulcerdisease, osteoporosis, aseptic (ischemic or avascular) bone necrosis,mental status changes, glaucoma, cataracts and hyperlipidemia (Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9^(th) edition,McGraw-Hill, NY, 1996, p.1475).

[0164] Induction of sTNFR1 shedding from cell culture systems can beinduced by a variety of physiological and non-physiological mediators,such as, IFN-γ, IL-1β, IL-6, formyl-Met-Leu-Phe (fMLP),lipopolysaccharide, 4b-phorbol 12-myristate 13-acetate (PMA), calciumionophore, staurosporine, and sodium salicylate (Porteu et al., J. Biol.Chem., 266:18846-18853 [1991]; Nicod. et al., Ann. NY Acad. Sci.,28:323-333 [1994]; Tilg et al., Blood 83:113-118 [1994]; Zhang et al.,J. Biol. Chem., 269:10270-10279 [1994]; Mullberg et al., J. Immunol.,155:5198-5205 [1995]; Levine et al., Am. J. Respir. Cell Mol. Biol.,14:254-261 [1996]; and Madge et al., J. Biol. Chem., 274:13643-13649[1999]). However, these agents are poor candidates for development asdrugs to regulate TNF activity in humans for a variety of reasons, mostnotably for the fact that most of these agents are toxic and notsuitable for human therapeutic use. In addition, most of these agentsare known to have or are likely to have detrimental or unwanted sideeffects resulting from their disruption of normal body processes inaddition to their effects on TNF signaling.

[0165] Other strategies for the regulation of TNF activity have alsobeen attempted or proposed. For example, U.S. Pat. Nos. 5,519,000 and5,641,751 to Heavner et al. (hereby incorporated by reference) describeshort (4-25 amino acid) peptides which bind TNF and inhibit TNFactivity. U.S. Pat. Nos. 5,665,859 and 5,766,917 to Wallach et al.(hereby incorporated by reference) describe methods to isolate a TNFR1protease or other polypeptides which influence TNFR1 shedding. U.S. Pat.No. 5,945,397 to Smith et al. (hereby incorporated by reference)describes human and mouse soluble TNF receptors and mutant variations ofthese receptors. U.S. Pat. No. 5,919,452 to Le et al. (herebyincorporated by reference) describes the use of anti-TNF antibodies,anti-TNF peptides, and soluble TNFR to treat TNF mediated pathologies.

[0166] However, until the development of the present invention, anucleic acid in the form of a cDNA and a polypeptide encoded by the cDNAwhich has the ability to regulate the shedding of the TNFR1 ectodomainfrom the extracellular surface of a cell plasma membrane to yield afree, soluble form of the receptor, sTNFR1 had not been described.Indeed, database searches revealed that this gene and polypeptide hadnot heretofore been identified. These searches also revealed amino acidsequence motifs indicative of peptidase activity. Specifically, thisgene has been named aminopeptidase regulator of type I, 55 kDa tumornecrosis factor receptor shedding, or “ARTS-1.” Methods for the use ofthe ARTS-1 gene, polypeptide and other related compositions are providedby the present invention. In addition, methods for the identification ofgenes and polypeptides substantially homologous to ARTS-1 gene andpolypeptide are also provided by the present invention. However, anunderstanding of the mechanisms of ARTS-1 activity is not necessary topractice the present invention.

[0167] It is contemplated that the protein (or proteins), and theircorresponding genes, which regulate the cleavage of TNFR1 from the cellsurface are likely to have important roles in regulating TNF activity invivo in health and disease states. It is contemplated that cleavage ofthe TNFR1 ectodomain from the cell surface limits TNF activity byproviding a pool of free receptors capable of binding and sequesteringTNF, as well as by removing functional TNFR1 from the cell surface. Itis further contemplated that occupation of the TNF bindings site on thefree sTNFR1 ectodomain does not activate the intracellular componentsinvolved in TNF signaling. It is further contemplated that the genes andproteins which regulate the shedding of soluble TNFR1 ectodomain willfind significant use in diagnostics and therapeutic regimens, as well asin the research setting. Furthermore, it is contemplated that genes andproteins which regulate the shedding of TNFR1 also regulate the sheddingof other cytokine receptors important in inflammatory diseases anddisorders (e.g., IL-1 and IL-6 receptors).

[0168] The present invention provides compositions and methods toidentify genes and proteins which regulate the cleavage and shedding ofthe TNFR1 ectodomain. In addition, the present invention providescompositions suitable for the production of monoclonal and/or polyclonalantibodies directed against the proteins involved in the shedding ofcytokine receptors (e.g., TNFR1). The present invention also providescompositions and methods suitable for the development of diagnostictools and assay systems for the assessment of the factors involved inregulation of soluble TNFR1as an indicator of health and/or disease.

[0169] The present invention also provides compositions and methodssuitable for the development of more effective therapies for treatingdiseases and disorders resulting from aberrant TNF activity (e.g., fordownregulation or upregulation of TNF activity), and consequentlyprovide therapeutic advantages for treatment of diseases or disordersresulting from inflammatory response or immune-deficiency.

[0170] In addition, the present invention provides compositions andmethods to develop additional means for suppressing damagingTNF-mediated proinflammatory diseases or disorders without the toxicside effects associated with existing techniques for immune suppression.The compositions and methods provided by the present invention addressthe need to identify genes and proteins which regulate TNF activity, andconsequently, have therapeutic value in the treatment of immunedisorders.

[0171] The remainder of the Description of the Invention is divided intothe following sections:

[0172] I) Cloning of Genes Encoding TNFR1Ectodomain Binding Polypeptides

[0173] II) Preparation of Recombinant Vectors and Transformants

[0174] III) Analysis of ARTS-1 mRNA Expression

[0175] IV) Antibodies Directed Against ARTS-1 Polypeptide

[0176] V) Detection of ARTS-1 Polypeptide in Cultured and Primary Cells

[0177] VI) GST-ARTS-1 Polypeptide Expression and Purification

[0178] VII) Analysis of ARTS-1 Polypeptide Aminopeptidase Activity

[0179] VIII) Analysis of ARTS-1 TNFR1 Ectodomain Sheddase RegulatoryActivity

[0180] IX) Analysis of TNFR1 Ectodomain Sheddase Regulatory Activity ofARTS-1 Catalytic Mutants

[0181] X) Demonstration of ARTS-1/TNFR1 Protein Interaction

[0182] XI) Therapeutic Agents for Immune Diseases and Disorders

[0183] XII) Diagnostic Agents for Immune Disease and Disorders

[0184] XIII) Identification of Genes Substantially Homologous to ARTS-1

[0185] XIV) Methods of Drug Screening

[0186] I. Cloning of Genes Encoding TNFR1 Ectodomain BindingPolypeptides

[0187] A) Yeast Two-Hybrid Screening

[0188] It was contemplated that polypeptides which form a physicalinteraction with the ectodomain of TNFR1 would be potential candidatesfor polypeptides which regulate the cleavage and shedding of the TNFR1ectodomain. A yeast two-hybrid screen was performed in order to identifysuch TNFR1 interacting polypeptides using a TNFR1 bait and human lungcDNA two-hybrid prey library. Yeast two-hybrid screening is a commontechnique in the molecular biology arts (Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, p. 20.0.1-20.1.40, John Wiley & Sons,Inc., New York [1994]). In the development of the present invention, theprotocols, yeast strains and reagents used in these experiments werethose supplied or recommended by the manufacturer (Matchmaker System 2;Yeastmaker Transformation System; Clontech), except where explicitlystated otherwise. The methods and compositions provided and used duringthe development of the present invention are provided in more detailbelow and in the Examples.

[0189] Conventional methods known to one of skill in the art may be usedto prepare mRNA to construct a yeast-two hybrid library. In general, thesource of the mRNA (e.g., tissue or cultured cells) are treated with aguanidine reagent, phenol reagent or the like to obtain the total RNA.Subsequently, poly(A+)RNA (i.e., mRNA) is obtained therefrom by anaffinity column method using oligo dT-cellulose or poly U-Sepharosecarried on Sepharose 2B. Further, the resultant poly(A+) RNA can befurther fractionated by sucrose gradient centrifugation or the like.

[0190] Single-stranded cDNA is synthesized using the thus obtained mRNAas a template, an oligo(dT) primer and a reverse transcriptase. Then, adouble-stranded cDNA is synthesized from the resultant single-strandedcDNA. The resultant double-stranded cDNA is integrated (i.e., ligated)into an appropriate cloning vector. In the case of this yeast two-hybridlibrary, the resulting double-stranded cDNAs are cloned into the pGAD10plasmid (Clontech), a recombinant vector which contains operably linkedparts which enable the transcription and translation of a chimeric geneconsisting of the transcriptional activation domain of the yeast GAL4transcription factor and the subcloned cDNA. The pGAD10 vector containsadditional operably linked parts which enable its selection, replicationand propagation in yeast strains as well as E. coli.

[0191] As indicated above, the yeast two-hybrid screen was conductedusing the manufacturer's recommended protocols. A bait vector wasconstructed using the pAS2-1 plasmid (Clontech), to produce a chimericfusion gene containing nucleic acid sequences encoding the DNA bindingdomain of GAL4 and the extracellular domain of the human TNFR1 receptor,corresponding to amino acids 26-216. Following sequential transformationof the chimeric pAS2-1 bait and pGAD10 prey vector library into yeaststrain Y190, the library was screened for clones positive for growth onhistidine (HIS) deficient media, followed by identification of thoseHIS⁺ clones which were also positive in a β-galactosidase filter liftassay. Thirty three clones positive for the ability to grown on HISdeficient substrate and β-galactosidase activity were identified, andeach of those clones was sequenced. It is noted that numerous protocolsand reagents for DNA sequencing are commercially available. It iscontemplated that any suitable method known in the art may readily beused in place of the protocol described here as well as other aspects ofthe production of the compositions of the present invention.

[0192] One positive clone, L26C-53A, was selected for further study.This clone contained a 2355 bp insert containing a 631 amino acid openreading frame with a consensus zinc metalloprotease catalytic motif. Thenucleotide sequence of this clone corresponds to bases 1044 to 3082 ofthe nucleic acid of FIG. 1 (SEQ ID NO:1). This gene was namedaminopeptidase regulator of type I, 55 kDa tumor necrosis factorreceptor ectodomain shedding, or “ARTS-1.”

[0193] The yeast two-hybrid screening described herein is not meant tolimit the present invention to the particular reagents and methodsdescribed. Indeed, numerous other vectors and reagents may be used toproduce the ARTS-1 gene provided by the present invention. For example,a different two-hybrid prey library may be substituted for the humanlung cDNA library to yield the ARTS-1 gene. Similarly, pAS2-1 and pGAD10vector variants may be used, for example those which have differentmultiple cloning sites to facilitate subcloning of the eDNA insert.Vector variants which use the LexA operator components in place of theGAL4 system may also be used in place of those described here. Also,variants of these vectors with or without an operably joined HAantigenic tag may be used in order to facilitate immunodetection of thefusion protein. Similarly, Saccharomyces cerevisiae strains other thanY190 may be used as the doubly transformed host strain for the libraryscreening (e.g., S. cerevisiae strain CG-1945). In addition, selectionconditions may be varied, for example, by changing the concentration of3-AT (3-amino-1,2,4-triazole) to counterselect leaky HIS⁺ clones. Manyof these alternative variables and reagents are described and arecommercially available (e.g., from companies such as Stratagene andClontech). Thus, it is intended that the compositions of the presentinvention may be produced using any suitable method known in the art.

[0194] B) Phage Plaque Hybridization/cDNA Cloning

[0195] The DNA sequence identified in the yeast interaction screenappeared not to be a full length cDNA. This, the complete ARTS-1 cDNAwas isolated by screening a phage library using a ³²P-labelled DNA probederived from clone L26C-53A. The library used in this cDNA screening wasconstructed using poly (A⁺) mRNA from the human NCI-H292 pulmonarymucoepidermoid carcinoma cell line (ATCC, CRL 1848) which had beenstimulated with 1 μM PMA (phorbol 12-myristate, 13-acetate, Sigma). Thiscell line has been demonstrated to shed sTNFR1 in response to PMAstimulation (Levine et al., Am. J. Respir. Cell. Mol. Biol., 14:254-261[1996]). The library was constructed using the uni-ZAP XR phage vector(Stratagene).

[0196] Bacteriophage from the library were plated in a lawn of XL1-BlueE. coli (Stratagene) at a density of 50,000 pfu per 150 mm plate andincubated overnight at 37° C. Plaques were transferred to Hybond N+filters (Amersham/Pharmacia) and denatured. Filters were thenneutralized and UV cross-linked. Filters were washed inpre-hybridization solution, then hybridized overnight at 42° C. with a³²P-labelled L26C-53A insert generated by random primed labelling.Filters were washed, and were then exposed to x-ray film overnight andpositive plaques were selected. Positive plaques were subjected to twoadditional rounds of plaque hybridization prior to sequencing. Positiveplaques were recovered via in vivo excision utilizing the ExAssisthelper phage (Stratagene).

[0197] It is not intended that the present invention be limited to anyparticular cDNA library, reagent or method for the production of thenucleic acid encoding the ARTS-1 polypeptide of the present invention.Thus, it is contemplated that any suitable library will find use inisolating the ARTS-1 cDNA. For example, such libraries may includelibraries made from mRNA derived from human chronic myelogenous leukemiacell line K-562, lymphoblastic leukemia cell line MOLT-4 or lungcarcinoma A549 cell line. Similarly, multiple techniques for theradiolabelling and purification of nucleic acid probes as well as phageplaque hybridization protocols are well known in the art (Ausubel et al.(eds.), Current Protocols in Molecular Biology, Vol. 1-4, John Wiley &Sons, Inc., New York [1994]). The probe synthesis and hybridizationmethods and conditions described here are not meant to limit the scopeof the present invention. Variations on these protocols includealternative labelling methods (e.g., 5′ end labelling, overhang fill-inlabelling and random primed synthesis labelling). Alternative detectionsystems can also be used, including ³³P probe labelling andnon-radioactive probe visualization methods such as chemiluminescence.

[0198] Following three rounds of screening, four hybridizing phageclones were identified from the library, amplified, then sequenced.These four clones all overlapped the L26C-53A sequence, as expected.However, none encoded a full-length cDNA, nor did they collectively spanthe entire gene. One phage clone (bp 1-1777) contained the putative 5′UTR and three phage clones contained the putative 3′ UTR and the poly(A)tail (bp 2181-4845). cDNA sequence encoding the portion of the genelying between the 5′ and 3′ terminal clones was amplified via PCR fromthe same human lung cDNA library using primers corresponding to 5′ and3′ sequence obtained from the phage screening. The cDNA segmentamplified with these primers was subcloned and both strands weresequenced to verify PCR fidelity.

[0199] As discussed in Section A above, it is not intended that thepresent invention be limited to any particular reagents, methods, and/orcompositions for the production of the nucleic acids and polypeptides ofthe present invention. Thus, it is contemplated that any suitablemethods for cDNA cloning and/or phage plaque hybridization will find usein the production of the compositions of the present invention. Forexample, one of numerous commercially available DNA polymerases may beused in the PCR reaction, including Taq (Stratagene, Promega), Pfu(Promega) or Sequenase (Amersham). Furthermore, it is contemplated thatalternative PCR reaction conditions (i.e., temperatures and timeintervals) will also be successful in amplifying the ARTS-1 nucleic acidof the present invention. Similarly, numerous vectors and reagents areequally suitable for subeloning and sequencing reactions.

[0200] C) Analysis of the ARTS-1 cDNA and Predicted Polypeptide

[0201] Inspection of the full length cDNA obtained as described aboverevealed a 4845 nucleotide transcript, containing a 2823 bp open readingframe and 5′ and 3′ untranslated regions as shown in FIG. 1. In thisFigure, a consensus polyadenylation site located at nucleotides 4795 to4800 is indicated with a double underline and lies 18 nucleotidesupstream of a 27 nucleotide poly(A) tail. Two mRNA destabilizationmotifs are also identified at nucleotides 3929 and 4457 using bold andunderline.

[0202] The open reading frame (ORF) encodes a 941 amino acidpolypeptide. The first ATG codon lying in-frame relative to the largestopen reading frame is located at nucleotide 88, and a TAA stop codon inthe same reading frame is located at nucleotide 2911. Asparagineresidues comprising five potential N-glycosylation sites are indicatedwith circles. A putative transmembrane domain, extending from aminoacids 5 to 28, is also indicated in the Figure with a single underline.In order to determine this sequence, sequence analysis, includingKyte-Doolittle hydropathy prediction (Kyte and Doolittle, J. Mol. Biol.,157:105-132 [1982]) was performed using MacVector 7.0 software (OxfordMolecular). The location of the putative hydrophobic transmembraneα-helical domain was predicted utilizing several web-based analysisprograms (MEMSAT2 (McGuffin et al., Bioinform., 16:404-405 [2000]; Sosui(Hirokawa et al., Bioinform., 14:378-379 [1998]; TMAP (Persson andArgos, J. Mol. Biol., 237:182-192 [1994]); TMpred (Hofmann and Stoffel,Biol. Chem. Hoppe-Seyler 374:166 [1993]; and TopPred2 (von Heijne, J.Mol. Biol., 225:487-494 [1992]). In sum, ARTS-1 is predicted to be atype II integral membrane protein with a single hydrophobictransmembrane α-helical domain, located between amino acids 5 and 28(See, FIGS. 1 and 2), and a very short hydrophobic intracellularamino-terminal domain (See, McGuffin et al., Bioinform., 16:404-405[2000]; Hirokawa et al., Bioinform., 14:378-379 [1998]; Persson andArgos, J. Mol. Biol., 237:182-192 [1994]); Hofinann and Stoffel, Biol.Chem. Hoppe-Seyler 374:166 [1993]; and von Heijne, J. Mol. Biol.,225:487-494 [1992]).

[0203] Subdomains of homology indicate that ARTS-1 is a member of theaminopeptidase family of gluzincin zinc metalloproteases. This family ofproteins share motifs indicating similar biochemical activity, althoughthe family members are extremely diverse in overall structure inbiological function (Zinc Metalloproteases in Health and Disease, Taylor& Francis, London, England [1996], Hooper, p.1-21, and Wang and Cooper,p.131-151). FIG. 1 also indicates the ARTS-1 sequences which adhere tothe consensus zinc metalloprotease catalytic motif for theaminopeptidase family, HEXXH(Y)₁₈E (SEQ ID NO:10). Within this motif, itis theorized that the two histidine residues (H) and the second glutamicacid residue (E) represent the zinc binding domain while the firstglutamic acid mediates the catalytic activity. In the ARTS-1polypeptide, this consensus motif is observed at T³⁵⁰VAHELAHQWFG (SEQ IDNO:8) and L³⁷²WLNEGFA (SEQ ID NO:9) (boxed in FIG. 1).

[0204] Furthermore, a schematic representation of the ARTS-1 protein,indicating domains of homology with the aminopeptidase family ofgluzincin zinc metalloproteases is provided in FIG. 2. A zincmetalloprotease consensus catalytic motif HEXXH(Y)₁₈E (SEQ ID NO:10), ashort intracytoplasmic tail, a transmembrane domain and a large 375amino acid domain of homology are also depicted in this Figure.

[0205] Quantitative comparisons between ARTS-1 protein and other membersof the aminopeptidase-gluzincin zinc metalloprotease family are shown inTables 1 and 2 below. Table 1 provides quantitation of percent identityand percent similarity between the full length amino acid sequence ofthe ARTS-1 protein and other aminopeptidase family members. Percentidentities are shown above the shaded diagonal and percent similaritiesare shown below the shaded diagonal. Table 2 shows a similar comparisonbetween the conserved 375 amino acid domain in the ARTS-1 proteincontaining the consensus zinc binding motif HEXXH(Y)₁₈E (SEQ ID NO:10)and other members of the aminopeptidase family of gluzincin zincmetalloproteases. The aminopeptidase family members included in theseTables are human placental leucine aminopeptidase (PLAP) (Rogi et al.,J. Biol. Chem., 271:56-61 [1996]), rat insulin-regulated aminopeptidase(IRAP) (Keller et al., J. Biol. Chem., 270:23612-23618 [1995]), humanaminopeptidase A (AMP A) (Nanus et al., Proc. Natl. Acad. Sci. USA90:7069-7073 [1993]; and Li et al., Genomics 17:657-664 [1993]), humanaminopeptidase N (AMP N) (Olsen et al., FEBS Lett., 238:307-314 [1988]),human puromycin sensitive aminopeptidase (Tobler et al., J. Neurochem.,68:889-897 [1997]), rat thyrotropin-releasing hormone degrading enzyme(TRH DE) (Schauder et al., Proc. Natl. Acad. Sci. USA 91:9534-9538[1994]), S. cerevisiae aminopeptidase YSCII (Garcia-Alvarez et al, Eur.J. Biochem., 202:993-1002 [1991]), C. elegans cosmid F49E8.3 geneproduct (Wilson et al., Nature 368:32-38 [1994]), and Lactococcus lactisaminopeptidase N (Tan et al., FEBS Lett., 306:9-16 [1992]).

[0206] As mentioned above, and as indicated in these Tables, althoughmembers of this aminopeptidase family share a similar biochemical motif,there is significant sequence divergence among family members, likelyindicating distinct and specialized biological functions. TABLE 1Percentage Identity and Similarity Between Full Length ARTS-1 Proteinand Other Members of the Aminopeptidase-Gluzincin Zinc MetalloproteaseFamily Human Human Rat Human Human Rat Human C. elegans L. Lactis S.cerevisiae Protein ARTS-1 PLAP IRAP AMP A AMP N TRH DE PSA F49E8.3 AMP NYSC II Human 44 41 31 29 27 30 26 24 27 ARTS-1 Human 17 79 30 29 27 2825 23 26 PLAP Rat 15 5 28 28 28 26 24 21 24 IRAP Human 20 19 18 34 29 3025 22 28 AMP A Human 18 18 18 18 22 29 27 24 26 AMP N Rat 17 16 17 17 1426 23 22 22 TRH DE Human 17 19 17 16 17 16 36 28 32 PSA C. elegans 18 1715 18 17 17 16 28 31 F49E8.3 L. lactis 16 16 15 17 15 14 16 18 27 AMP NS. cerevisiae 16 18 16 15 16 16 16 19 18 YSC II

[0207] TABLE 2 Percentage Identity and Similarity Between the ConservedDomains Containing the Zinc Binding Motif of ARTS-1 Protein and OtherMembers of the Aminopeptidase-Gluzincin Zinc Metalloprotease FamilyAmino Acid Human Human Rat Human Human Rat Human C. elegans L. lactis S.cerevisiae Protein Position ARTS-1 PLAP IRAP AMP A AMP N TRH DE PSAF49E8.3 AMP N YSC II Human 161-535 51 53 39 43 41 44 42 36 41 ARTS-1Human 192-540 15 90 42 43 39 44 44 36 44 PLAP Rat 273-621 12 6 41 42 3944 45 37 43 IRAP Human 202-549 21 18 18 44 41 48 44 36 45 AMF A Human192-554 15 15 16 19 42 47 44 37 45 AMP N Rat 248-603 14 17 17 17 16 4140 37 38 TRH DE Human 158-508 16 18 17 14 14 18 52 41 52 PSA C. elgens120-469 16 17 16 17 17 20 16 41 49 F49E8.3 L. Lactis  99-445 16 20 18 1817 15 18 17 40 AMP N S. cerevisiae 114-461 15 16 15 16 16 19 15 18 20YSC II

[0208] II. Preparation of Recombinant Vectors and Transformants

[0209] Preparation of Recombinant Vectors

[0210] Numerous recombinant vectors relevant to the present inventionare contemplated. Such vectors, containing the gene or portions of thegene of the present invention (i.e., the ARTS-1 gene) may be of anytype, with the only limitation being that the vector into which the geneof the invention has been inserted has the capacity for replication in ahost. As used in the present invention, a host may be a bacteria (e.g.,E. coli BL21 strain), a yeast (e.g., S. cerevisiae Y190 strain), or ananimal cell (e.g., the NCI-H292 human pulmonary mucoepidermoid carcinomacell line). Furthermore, the host cell may exist in an in vitro tissueculture system, or exist within an intact organism (e.g., a mouse or ahuman). Indeed, it is not intended that the host be limited to anyparticular cell type, nor is it intended that the cell host bemaintained in any particular setting.

[0211] Protocols for the construction of recombinant vectors arecommonplace in the molecular biology arts, and reagents for themanipulation of recombinant nucleic acids are readily available fromcommercial sources. Routine procedures in the construction of arecombinant vector encompassed by the present invention may includerestriction endonuclease digestion, ligation, phosphorylation,dephosphorylation, blunt-ending, size separation, annealing, eluting,staining, desalting, transformation, inoculating, incubating, cesiumbanding and purification by a variety of commercially availableproducts. In the most simple of embodiments, a recombinant vector of theinvention may be made by digesting the gene of the invention with anappropriate restriction enzyme, ligating into an appropriately digestedvector, transforming the ligated vector into bacteria, selection usingan antibiotic resistance marker contained on the vector and purifyingthe vector using a kit designed for rapid, small scale plasmidpurification.

[0212] A vector may be in the form of a DNA plasmid. Such plasmidscontain multiple parts operably arranged to permit replication in aminimum of one specific host species. A plasmid (e.g., the pGEX-6P-1plasmid) may be restricted to replication in bacteria, or may alsocontain operably linked sequences which permit the plasmid to propagateor function in a second species in addition to its usual bacterial host.For example, the pAS2-1 and pGAD10 vectors contain operably linkednucleic acid sequences which permit their propagation in both bacteriaand yeast (e.g., the S. cerevisiae Y190 strain), while the pTargetvector contains operably linked sequences which permit its propagationin a bacterial host, but also contains sequences which allow it todirect the transcription of a cloned gene in a mammalian cell.

[0213] A vector may also be in the form of a DNA or RNA bacteriophage orother virus, which further may be in the form of a DNA or RNA containingvirus. Such phage or other virus may be replication competent orreplication defective. The phage or viral nucleic acid may also beengineered to permit propagation in an organism as a plasmid in additionto its viral life cycle. For example, the bacteriophage Lambda (λ)uni-ZAP II vector (Stratagene) is capable of bacterial infection as abacteriophage, but also contains sequences which enable the excision andpropagation of a plasmid form of the vector. Such a vector is oftencalled a “phagemid.” Further, recombinant viral vectors (e.g.,retrovirus, adenovirus, adeno-associated virus or vaccinia virus) or aninsect virus vector (e.g., baculovirus) may also be used to infectcells.

[0214] In particularly preferred embodiments, the gene of the presentinvention is operably linked to other components of the vector. For thispurpose, the vector of the invention may contain, if desired, ciselements (e.g., an enhancer, splicing signal, poly(A) addition signal,selection marker, ribosome binding sequence (i.e., Shine-Dalgarno orKozak sequences) or the like) in addition to a promoter/enhancer and thegene of the present invention. As the selection marker, genes encodingresistance to dihydrofolate reductase, ampicillin, kanamycin, neomycin,or the like may be used.

[0215] Preparation of Transformants

[0216] In some embodiments of the present invention, a transformant wasobtained by introducing the recombinant vector containing the gene ofthe invention into a host. The host is not particularly limited as longas it can harbor the vector of the invention. Specific examples of hostinclude Escherichia bacteria such as E. coli; yeast such as S.cerevisiae and Schizosaccharomyces pombe; animal cells such as COScells, CHO cells, NCI-H292 cells or non-specified cells of an intactorganism; or insect cells such as SF9 and SF21 cells. In someembodiments of the present invention, the vector is introduced into thehost under conditions such that the polypeptide encoded by the gene ofthe invention is expressed.

[0217] In some cases, when a bacterium such as E. coli is used as thehost, the recombinant vector of the invention is capable of replicationin the host and, at the same time, is capable of expressing the gene ofthe invention within the bacterial host (e.g. the pGEX-ARTS-1 vectors).Such vectors in general preferably consist of a promoter, a ribosomebinding sequence, the gene of the present invention and a transcriptiontermination sequence. The vector may also contain a gene to control thepromoter.

[0218] Any promoter may be used as long as it can appropriately directthe expression of the gene of interest in the host cells, such as amammalian cell or E. coli. For example, an E. coli or phage-derivedpromoter such as trp promoter, lac promoter, P_(L) promoter or P_(R)promoter may be used. An artificially designed and altered promoter suchas tac promoter may also be used.

[0219] Any method for bacterial transformation may be used forintroducing a recombinant vector into a bacterium. Many methods arecommonly known in the art, including electroporation and the use ofbacteria made competent by calcium chloride (See e.g., Ausubel et al.(eds.), Current Protocols in Molecular Biology, p. 1.8.1-1.8.10, JohnWiley & Sons, Inc., New York [1994]).

[0220] Likewise, any method for introducing DNA into yeast may be usedto introduce a recombinant vector into a yeast. For example,electroporation (Becker et al., Methods Enzymol., 194:182-187 [1991]),the spheroplast method (Hinnen et al., Proc. Natl. Acad. Sci. USA75:1929-1933 [1978]), the lithium acetate method (Ito, J. Bacteriol.,153:163-168 [1983]) or the like may be used.

[0221] It is contemplated that any suitable animal cells will find useas hosts in the present invention (e.g., simian COS-7 or Vero cells,Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3 cells,human FL cells, human lymphoid cell lines, or the like). Apromoter/enhancer may be used to express the ARTS-1 gene where thepromoter is active in all or most cell types (e.g., the SRα promoter,SV40 promoter, HSV-LTR promoter, CMV promoter or the like).Alternatively, promoter/enhancer elements may be used which directexpression of the ARTS-1 gene in only a subset of cells (e.g., cells ofthe lymphoid system).

[0222] As with other host cells, any suitable method for introducing therecombinant vector into animal or insect cells (e.g., electroporation,the calcium phosphate method, lipofection or the like) may be used.

[0223] III. Analysis of ARTS-1 mRNA Expression

[0224] Following the identification and isolation of the ARTS-1 gene, anexperiment was undertaken to determine the pattern of tissue expressionof the endogenous ARTS-1 mRNA. This was accomplished using amulti-tissue Northern blot and an ARTS-1 derived probe as shown in FIG.3 and described in greater detail in Example 3.

[0225] The blot used in this experiment was a commercially availablehuman multiple tissue poly(A+) Northern blot (Clontech). This blot wasprobed according to the manufacturer's suggested protocol using a fulllength ³²P-labelled ARTS-1 cDNA probe.

[0226] The Northern blot analysis shown in FIG. 3, Panel A, indicatesthat the human ARTS-1 transcript was expressed in multiple tissues,including spleen, thymus, small and large intestine, peripheral bloodleukocyte, heart, placenta, lung, skeletal muscle, kidney and pancreas.In these tissues, a single predominant mRNA species of approximately 5.7kB was detected. FIG. 3, Panel B, shows the same blot followingstripping and rehybridization to a probe specific for the human GAPDHtranscript as a reference for RNA loading normalization.

[0227] Furthermore, in view of numerous alternative protocols known inthe art for Northern blotting, it is not intended that the presentinvention be limited to the Northern blotting protocol provided inExample 3 or any other particular Northern blotting method. For example,in some embodiments, RNA is isolated from tissue samples usingalternative methods (e.g., a commercial RNA isolation kit such as QiagenRNeasy Total RNA Mini Kit, Catalog No. 74103).

[0228] Similarly, alternative probe synthesis and labelling techniquesalso find use with the present invention. For example, any probe havinga minimum complementarity of 25 base pairs to the ARTS-1 cDNA will finduse in the Northern blot methods of the present invention. Furthermore,it is contemplated that the nucleic acid comprising the probe will begenerated by PCR, by restriction digest, or by synthetic oligonucleotidesynthesis. Alternative nucleic acid probe labelling methods also finduse with the present invention (e g., labelling with ³³P radioisotope ornon-radioactive labelling methods). In addition, alternative Northernblotting protocols, reagents and equipment suitable for use in thepresent invention are known in the art (See, e.g., Ausubel et al.(eds.), Current Protocols in Molecular Biology, Vol. 1, pages4.9.1-4.9.16, John Wiley & Sons, Inc., New York [1994]).

[0229] IV. Antibodies Directed against the ARTS-1 Polypeptide

[0230] In the present invention, polyclonal antiserum directed againstthe ARTS-1 polypeptide is provided. However, the antibody of theinvention may be prepared by various methods, as numerous methods forthe production of monoclonal and polyclonal antibodies are well known inthe art (See e.g., Sambrook, J. et al. (eds.), Molecular Cloning, ColdSpring Harbor Laboratory Press [1989]; Harlow and Lane (eds.),Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press[1988]; Ausubel et al. (eds.), Current Protocols in Molecular Biology,p. 11.4.2-11.15.4, John Wiley & Sons, Inc., New York [1994]). As usedherein, the term “antibody” means an antibody molecule as a whole or afragment of an antibody (e.g., Fab or F(ab′)₂ fragment) which can bindspecifically an antigen. The antibody provided by the invention isdescribed in more detail below and in Example 4. The present inventionalso contemplates that these same methods will find use in theproduction of antibodies specific for polypeptides encoded by geneswhich are substantially homologous to the ARTS-1 gene.

[0231] In order to conduct additional studies on the ARTS-1 polypeptide,polyclonal antiserum was generated against an ARTS-1 polypeptide. Acommercial service (Research Genetics) was used in these studies.Specifically, a 17 amino acid ARTS-1 synthetic peptide was used toimmunize New Zealand white rabbits. This peptide corresponded to aminoacids 538 to 554 of the ARTS-1 polypeptide and had the sequence:

[0232] R⁵³⁸GRNVHMKQEHYMKGSD (SEQ ID NO:7)

[0233] This particular peptide was chosen based upon its antigenicpotential and its lack of homology with other protein sequences asdetermined by a BLAST homology search.

[0234] Rabbits were immunized with this peptide using standardtechniques. The ARTS-1 peptide was conjugated to KLH and mixed with anequal volume of Freund's complete adjuvant. The amount of antigenutilized per immunization was 0.1 mg, which was injected into threesubcutaneous dorsal sites. The animals received boosts at weeks 2, 6 and8. Bleeds were obtained at weeks 4, 8 and 10, and tested for thepresence of anti-ARTS-1 antibody. In subsequent experiments, theantiserum obtained from the 10 week bleed was used.

[0235] In view of numerous alternative protocols known in the art forthe production of polyclonal antibodies, the present invention is notmeant to be limited to any particular method. For example, the entire941 amino acid ARTS-1 polypeptide, or any portion or fragment thereof,may potentially be used as the immunogen, and the immunogen may beeither synthetic or native. It is not intended that the presentinvention be limited to any particular ARTS-1 derived immunogen, methodof immunization, immunization schedule, animal species, test protocolfor determining antibody production or antibody purification method.

[0236] Although the antibody provided by the invention is polyclonal,the invention also contemplates monoclonal antibodies directed againstthe ARTS-1 polypeptide. It is also contemplated that any suitable methodfor the production of monoclonal antibodies will find equal use in thepresent invention. In one embodiment, the immunogen used to producethese monoclonal antibodies comprises full-length ARTS-1 polypeptide,although it is contemplated that any portion or fragment of the ARTS-1polypeptide may also find use in the present invention as an imnmunogen.

[0237] In these monoclonal antibody protocols, any suitable method forrecovery of antibody producing cells, cell fusion, selection and cloningof hybridomas, recovery of the monoclonal antibodies, and purificationof the monoclonal antibodies of interest may be used. Thus, it is notintended that the present invention be limited to any particularmonoclonal antibody production system or method.

[0238] If desired, the polyclonal or monoclonal antibody preparation ofthe invention can be purified from crude antiserum or culturesupernatant using a conventional method (e.g., Protein A affinity,ammonium sulfate precipitation, ion exchange chromatography, gelfiltration, affinity chromatography, or any of these methods incombination).

[0239] Once the polyclonal or monoclonal antibody is thus obtained, insome embodiments of the present invention, the antigen is bound to asolid support, so as to thereby prepare an affinity chromatographycolumn. Using this column, the antibody of the present invention can behighly purified. Conversely, in an alternative embodiment of the presentinvention, the antibody is bound to a solid support as to therebyprepare an affinity chromatography column. Using this column, the ARTS-1polypeptide, or fragments thereof, either native or recombinant, can behighly purified from variable sources. These monoclonal and polyclonalantibodies find numerous uses, including Western blotting,immunoprecipitation, immunohistochemistry and clinical applicationsusing methods known in the art (See e.g., Harlow and Lane (eds.),Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press[1988]; Ausubel et al. (eds.), Current Protocols in Molecular Biology,Vol. 1-4, John Wiley & Sons, Inc., New York [1994]; and Laurino et al.,Ann. Clin. Lab Sci., 29(3):158-166 [19991]).

[0240] V. Detection of ARTS-1 Polypeptide in Cultured Cell Lines andPrimary Cells

[0241] Following the production of ARTS-1 polyclonal antiserum, theexpression of endogenous ARTS-1 polypeptide in cultured and primarycells was investigated using standard Western immunoblotting techniqueswell known in the art, as discussed below and described in furtherdetail in Example 5. Protein concentrations of the samples were assayedand 20 micrograms of total protein were prepared for analysis in Laemmlibuffer. Samples were resolved via 6% polyacrylamide SDS-PAGE andelectroblotted onto nitrocellulose. Blots were incubated overnight inblocking buffer, then incubated for 2 hours with ARTS-1 antiserum at a1:20,000 dilution in blocking buffer. Membranes were washed, thenincubated with horseradish peroxidase conjugated goat anti-rabbit IgG(Life Technologies) diluted to 1:5,000 in blocking buffer, then washedagain. Membranes were then incubated in chemiluminescent detectionsubstrate and the signal detected on X-ray filn.

[0242] The specificity of the resulting polyclonal antiserum was firstdetermined as shown in FIG. 4. Extracts analyzed in this experiment werecrude whole cell homogenates, and membrane and cytosolic fractions allprepared from cultured NCI-H292 cells. As shown in FIG. 4, Panel A, theanti-ARTS-1 antiserum detected a predominant 100 kDa membrane form and apredominant 68 kDa cytosolic form from the NCI-H292 cells, while thewhole cell extracts predictably revealed a mixture of these two forms.As shown in FIG. 4, Panel B, the preimmune serum showed no reactivitytowards the same samples when used at the same concentration.

[0243] Specificity of the immune serum was further demonstrated incompetition experiments in FIG. 4, Panels C and D. Preincubation of theimmune serum with the RGRNVHMKQEHYMKGSD peptide (SEQ ID NO:7) againstwhich the polyclonal antiserum was raised resulted in almost completeattenuation of the immune signal (Panel D). In contrast, preincubationof the immune serum with bovine serum albumin resulted in minimalattenuation of immune signal (Panel C).

[0244] The expression of endogenous ARTS-1 polypeptide in primary cellsand other cell lines was further investigated using the identicalantiserum and Western immunoblot technique (See, FIG. 5). Theseexperiments were conducted to determine if there were differences in theARTS-1 polypeptide forms expressed in different cell lines. Theseexperiments analyzed membrane and cytosolic fractions made from humanbronchial brushing specimens, airway epithelial cell lines BEAS-2B andBET-1A, human lung carcinoma cell line A549, cultured NCI-H292 cells,primary cultures of normal human bronchial epithelial cells (NHBE),human umbilical vein endothelial cells (HUVEC) and human fibroblasts.

[0245] Similar to the NCI-H292 cell line, the BEAS-2B, BET-1A, NHBE andA549 cell lines all revealed a 100 kDa band localized predominantly inthe membrane fraction, and a 68 kDa form expressed to varying degrees inthe cytosolic fraction. The protein samples obtained from humanbronchial brushings, as well as the HUVEC and fibroblast primary cellsrevealed similar patterns, with the exception that both the 100 and 68kDa forms appear in the membrane fraction. The human bronchial brushcells and occasionally the NCI-H292 cell line also showed a larger 132kDa form.

[0246] These multiple sized forms may be due to regulated processing ofthe ARTS-1 polypeptide. Furthermore, the distinction between thedifferent sized ARTS-1 forms seen in the membrane versus the cytosolicfractions may also indicate regulated processing between the membraneand cytosol. However, it is not necessary to understand the mechanism ofARTS-1 processing or localization in order to practice the presentinvention, nor is it intended that the present invention be so limited.

[0247] Furthermore, in view of numerous alternative protocols known inthe art for Western blotting, it is not intended that the presentinvention be limited to the Western blotting protocol provided inExample 5 or any other Western blotting method. For example, in someembodiments, alternative secondary (i.e., detection) antibodies can beused. Alternative Western irmnunoblotting protocols, reagents andequipment suitable for use in the present invention are known in the art(See, e.g., Ausubel et al. (eds.), Current Protocols in MolecularBiology, Section 10.8, “Immunoblotting and Inmmunodetection,” John Wiley& Sons, Inc., New York [1994]).

[0248] VI. GST-ARTS-1 Polypeptide Expression and Purification

[0249] In order to facilitate the analysis of ARTS-1 biochemicalactivity, a highly purified form of the ARTS-1 polypeptide was producedusing a GST fusion protein protocol commonly used in the art. Thespecifics of this protocol are provided in detail in Exarnple 6. It isnot intended that the present invention be limited to any particularmethod for GST-ARTS-1 polypeptide expression, either with or withoutsubsequent GST-ARTS-1 polypeptide purification.

[0250] The use of GST fusion proteins to produce purified polypeptidesis common in the art. In these protocols, a transcriptional andtranslational fusion is made between the genes encodingglutathione-S-transferase (GST) and the gene of interest (e.g. theARTS-1 gene). Production of the fusion protein containing a GST “tag”enables the effective and rapid purification of the fusion protein byuse of an affinity column comprising immobilized glutathione. In orderto produce a GST-ARTS-1 polypeptide, the cDNA sequence encoding ARTS-1was first PCR amplified and subeloned into the pGEX plasmid backbone(Amersham Pharmacia) and used to transform the BL21 E. coli host strain.The pGEX plasmid contains a multiple cloning site and expresses a fusionprotein consisting of the GST polypeptide and a subdloned codingsequence (e.g., the ARTS-1 coding sequence) cloned into the multiplecloning site of the plasmid. Transcription of the fusion gene iscontrolled by the conditional tac promoter. In accordance with themanufacturer's protocol, clones were cultured in the presence of 0.6 mMisopropyl β-D-thiogalactoside (IPTG) and subsequently lysed with aprotein extraction buffer. Transformed clones expressing GST-ARTS-1fusion protein were selected by Western immunoblotting utilizing ananti-GST antibody (Amersham Pharmacia). Cells with confirmed expressionof GST-ARTS-1 polypeptide were lysed and centrifuged to separate thesoluble from the insoluble fractions. The GST-ARTS-1 fusion protein wasisolated from the insoluble fraction by denaturation with 6M urea, thendesalted and renatured. The GST-ARTS-1 fusion protein was purifiedutilizing a glutathione sepharose 4B affinity column using techniqueswell known in the art.

[0251] To assess the purity of the eluted recombinant GST-ARTS-1 fusionprotein, samples were subjected to SDS-PAGE and stained with Coomassiebrilliant blue, as shown in FIG. 6A. In this Figure, soluble andinsoluble protein fractions from BL21 E. coli transformed with emptypGEX vector are shown in lanes 1-2, and analogous samples from bacteriacontaining the pGEX-ARTS-1 vector are shown in lanes 3-4. The GST-ARTS-1fusion protein following elution is shown in lane 5, as a predominant132 kDa band, corresponding to the predicted molecular weight of aGST-ARTS-1 fusion protein (104 kDa ARTS-1 extracellular domain plus 26kDa GST tag). The control purified GST tag was revealed as a predicted26 kDa band in lane 6.

[0252] The recombinant purified GST-ARTS-1 fusion protein samples werefurther subjected to FPLC analysis to assess their purity, the resultsof which are shown in FIG. 6B. Elution from the FPLC column wasmonitored by absorbance at a wavelength of 280 nm. This analysis of thepurified GST-ARTS-1 revealed a single major protein elution peak atapproximately 40 minutes.

[0253] Using the aminopeptidase activity assay described below, FPLCfractions were assessed for aminopeptidase activity utilizing aphenylalanine p-nitroaniline substrate. The results of these experimentsare shown in FIG. 6C. As indicated in this Figure, the single majorprotein peak revealed by FPLC analysis coeluted with a peak ofaminopeptidase activity against a phenylalanine-pNA substrate in pooledfractions from 38-44 minutes.

[0254] As those of skill in the art know, numerous protocols for thepurification of polypeptides are available. The use of numerousalternative protocols for the production of isolated ARTS-1 polypeptideare easily envisioned. These alternative protocols may be used to purifyARTS-1 polypeptides other than the GST-ARTS-1 polypeptide described bythe present example. Indeed, it is not intended that the presentinvention be limited to any particular protocol.

[0255] Numerous reagents and variables may be manipulated or substitutedfor those used in Example 6 to yield a substantially similar purifiedARTS-1 polypeptide. Such variables include the choice of ARTS-1polypeptide (e.g., either with or without a fusion protein tag), thecells used for the production of the starting materials, thepromoters/enhancers used to drive expression of the recombinant proteinto be purified, the cellular culture and growth conditions, harvestingmethods, mode of purification, and methods to assess polypeptide purityfollowing purification. The detailed protocol provided in Example 6 isnot meant to limit the scope of the present invention. Indeed, it iscontemplated that any protocol which will produce a substantiallysimilar purified product will find use with the present invention. Suchalternative embodiments are briefly discussed below.

[0256] Alternative protocols may be used to purify forms of ARTS-1polypeptide other than the GST-ARTS-1 fusion polypeptide provided by thepresent invention. These alternative forms may include a maltose bindingprotein (MBP) ARTS-1 fusion polypeptide, a polyhistidine (i.e., 6×His)tagged ARTS-1 fusion polypeptide, a thioredoxin tagged ARTS-1 fusionpolypeptide, or a full length ARTS-1 polypeptide without any fused tagto facilitate purification.

[0257] Alternative protocols may find use in the present invention. Forexample, protocols which use different host systems as a source forstarting material (i.e., a source culture) for ARTS-1 purification maybe used. Such alternative source systems include insect cells with abaculovirus overexpression system (e.g., Sf9 or Sf21 cell lines),mammalian cell lines in conjunction with vectors designed forrecombinant polypeptide overexpression (expression vectors), ormammalian cells or tissues for the purification of ARTS-1 polypeptideexpressed from its endogenous (i.e., native) chromosomal location. Thecultivation of the transformed, transfected or infected host of theinvention is carried out in a medium and under conditions mostappropriate for the growth of that particular host cell. These mediaformulations and culture conditions are well known to those in the art.For example, culture of a mammalian cell line for the isolation of anoverexpressed or endogenous polypeptide will commonly use RPMI 1640 orDMEM media, typically supplemented with 5-10% fetal or newborn calfserum, and may be further supplemented with an antibiotic such askanamycin or penicillin. In some protocols, calf serum may be omitted tofacilitate subsequent polypeptide purification. Typically, thecultivation of mammalian cells is carried out in the presence 5% CO₂ at37° C.

[0258] Following the cultivation of a particular ARTS-1polypeptide-containing host, the proteins of the culture can beextracted by disrupting (by any suitable method) the microorganisms orcells if the protein is produced within the host. If ARTS-1 polypeptideof the invention is secreted by the host cells, the culture fluid iscollected. The resultant culture extract or culture supernatant is thensubjected to conventional biochemical techniques used for proteinpurification. As known in the art, numerous techniques for polypeptidepurification exist (e.g., ammonium sulfate precipitation, gelchromatography, ion exchange chromatography, and affinitychromatography). These techniques may be used independently or in anappropriate combination to isolate and purify the polypeptide(s) of thepresent invention from a culture.

[0259] VII. Analysis of ARTS-1 Polypeptide Aminopeptidase Activity

[0260] In light of the homology between the polypeptide predicted fromARTS-1 gene with aminopeptidase members of the gluzincin zincmetalloprotease family, it was determined if ARTS-1 polypeptide hadaminopeptidase activity, and furthermore, if the terminal aminopeptidaseactivity was specific for a particular amino acid or group of aminoacids.

[0261] To accomplish this, commercially available amino acidp-nitroanilide substrates were incubated with purified GST-ARTS-1 fusionprotein for 1 hour under conditions of linear enzyme activity over time.The rate of amide bond hydrolysis was determined by measuring theabsorbance of the p-nitroaniline aminopeptidase reaction product at 380nm. Each experimental point was run in triplicate and each determinationutilized six concentrations of amino acid p-nitroanilide substrate.Kinetic constants were determined by Lineweaver-Burk analysis.Correlation coefficients for each line generated were greater than0.997. The results of this analysis are shown in Table 3 below. Eachamino acid p-nitroaniline substrate tested is shown in the left colurnr,along with its polar/non-polar/acidic/basic nature. V_(max) is a measureof the theoretical enzyme maximal velocity, K_(m) is theMichaelis-Menten constant for each ARTS-1/substrate combination, k_(cat)is the first order rate constant (i.e., the turnover number) for theARTS-1/substrate combination, and k_(cat)/K_(m) is the second order rateconstant for the ARTS-1/substrate combination (i.e., a measure ofoverall catalytic efficiency).

[0262] As shown in Table 3, recombinant GST-ARTS-1 protein possessedselective aminopeptidase activity against non-polar amino acidsubstrates with a four-fold range of enzyme activity. Isoleucine-pNA wasfound to be the most favorable amino acid substrate based uponk_(cat)/K_(m) determination, followed byPhe>Gly>Cys>Leu>Met>Ala>Pro>Val. Recombinant GST-ARTS-1 had no activityagainst either acidic (Asp or Glu) or basic (Arg, His, or Lys) aminoacid substrates. TABLE 3 Specificity and Rates of ARTS-1 AminopeptidaseActivity V_(max) aa-pNA Polarity (pmol/pmol/min) K_(m) (mM) k_(cat)(s⁻¹) × 10⁻² k_(cat)/K_(m) (s⁻¹M⁻¹) Ile Non-polar 5.81 ± 0.87 1.67 ±0.02 9.68 ± 0.15 57.98 Phe Non-polar 5.14 ± 0.04 1.66 ± 0.03 8.57 ± 0.0651.61 Gly Non-polar 8.67 ± 0.05 3.67 ± 0.03 14.45 ± 0.08 39.37 CysNon-polar 8.95 ± 0.31 4.57 ± 0.20 14.92 ± 0.52 32.64 Leu Non-polar 9.45± 0.43 5.26 ± 0.25 15.75 ± 0.72 29.94 Met Non-polar 13.36 ± 0.75  7.71 ±0.43 22.27 ± 1.25 28.88 Ala Non-polar 26.18 ± 0.24  16.84 ± 0.18  43.63± 0.40  25.91 Pro Non-polar 5.29 ± 0.08 4.68 ± 0.06 8.82 ± 0.13 18.84Val Non-polar 5.31 ± 0.31 5.69 ± 0.26 8.85 ± 0.52 15.50 Asp Acidic NoActivity — No Activity — Glu Acidic No Activity — No Activity — ArgBasic No Activity — No Activity — His Basic No Activity — No Activity —Lys Basic No Activity — No Activity —

[0263] VIII. Analysis of ARTS-1 TNFR1 Ectodomain Sheddase RegulatoryActivity

[0264] In light of the ability of the ARTS-1 polypeptide to bind to theTNFR1 ectodomain in the yeast two-hybrid interaction assay and thepeptidase/protease motif contained within the predicted polypeptide, theability of ARTS-1 to promote the shedding of the TNFR1 ectodomain fromthe surface of human cells in culture was examined. The methods used inthis experiment are described in detail in Examples 9 and 11. Theresults are depicted in FIGS. 7 and 8.

[0265] This experiment was done in two phases. The first phase involvedconstruction of stably transfected cell lines which expressed eitherreduced or elevated levels of ARTS-1 polypeptide, as detailed in Example9. The NCI-H292 cell line was stably transfected with one of threeconstructs, all based on the pTarget vector. The pTarget vector containselements which enable its selection following stable integration in theNCI-H292 cell line, and also has the ability to constitutively express acloned insert in mammalian cells. The pTarget vectors used in thisexperiment were:

[0266] 1) an empty pTarget vector,

[0267] 2) pTarget vector containing the fuill length ARTS-1 cDNA codingregion in the sense orientation,

[0268] 3) pTarget vector containing ARTS-1 cDNA bases 61 to 213 in theanti-sense orientation (this region overlaps the putative transcriptionstart site and intracellular and transmembrane domains).

[0269] Following the introduction and selection of these constructs inthe host cell lines, membrane fractions were prepared from the lines andsubject to Western immunoblotting in order to assess ARTS-1 polypeptideexpression. This analysis was conducted according to the Westernimmunoblotting technique described in Example 5 and used the anti-ARTS-1polyclonal antiserum produced as described in Example 4. The results ofthis analysis are shown in FIG. 7. In that Figure, two independentclonal lines containing ARTS-1 sense or antisense expressing vectorswere analyzed. It was found that integration of the empty pTarget vector(Mock) had little effect on endogenous ARTS-1 expression compared tocells that did not contain any stably integrated plasmid (WT). The celllines expressing the fuill length ARTS-1 cDNA in the sense orientation(ARTS-1) showed a significant increase in ARTS-1 protein expression,while the cell lines expressing the ARTS-1 antisense sequence (AS)showed significant reduction in ARTS-1 protein expression.

[0270] The amount of TNFR1 ectodomain shedding occurring in each ofthese cell lines is depicted in FIG. 8. The levels of sTNFR1 ectodomainin cell culture supernatants from these cell lines were assayed using acommercially available sandwich-enzyme-linked immunosorbent assay(ELISA) technique (R & D Systems) with a lower limit of detection of 7.8pg/ml. In FIG. 8, results are displayed as the mean of 5 independentexperiments, with accompanying SEM (standard error of the mean). Asshown in the Figure, the cell lines showing increased ARTS-1 proteinexpression (ARTS-1) also showed a significant increase in the amount ofsTNFR1 present in cell culture supernatants as compared to cellstransfected with the empty pTarget vector (Mock). Conversely, cell lineswith decreased ARTS-1 protein expression (AS) showed significantlydecreased levels of sTNFR1 in cell culture supernatants as compared tocells transfected with the empty pTarget vector (Mock).

[0271] The degree of TNFR1 ectodomain shedding as a function of ARTS-1protein expression was also assessed indirectly by determining therelative amounts of membrane-bound TNFR1 fragment in each of the stablytransfected cell lines described above using the Western immunoblottingtechnique described in Example 5. Crude membrane fractions from thestably transfected NCI-H292 cells described in Example 9 were preparedand resolved by SDS-PAGE and analyzed by Western immunoblotting using amurine anti-human TNFR1 monoclonal primary antibody (R & D System) whichdetects the membrane fragment of the TNF receptor. This Western blot isshown in FIG. 11. As shown in FIG. 11, cell lines over-expressing ARTS-1(ARTS-1) demonstrated a decrease in membrane-associated TNFR1 relativeto non-transfected (WT) or control transfected (Mock) cell lines,consistent with an increase in constitutive TNFR1 ectodomain shedding.Conversely, cell lines expressing anti-sense ARTS-1 mRNA (AS)demonstrated an increase in membrane-associated TNFR1 relative tonon-transfected (WT) or control transfected (Mock) cell lines,consistent with a reduction in constitutive TNFR1 ectodomain shedding.

[0272] Results from an experiment analyzing the ability of ARTS-1overexpression to potentiate the shedding of TNFR ectodomain from thesurface of NCI-H292 cells in response to PMA stimulation using thesesame cell lines are shown in FIG. 9. Cell lines overexpressing fulllength ARTS-1 mRNA were stimulated with 0.1 μM phorbol 12-myristate13-acetate (PMA), which has previously been shown to upregulate sTNFR1shedding in NCI-H292 cells (Levine et al., Am. J. Respir. Cell Mol.Biol., 14:254-261 [1996]). As indicated in FIG. 9, the cell linecontaining only the empty pTarget vector showed only a modest increasein sTNFR1 shedding following 24 hours of PMA treatment. However, thecell line overexpressing the ARTS-1 cDNA showed a more dramatic increasein sTNFR1 shedding following 24 hours of PMA treatment, increasing fromapproximately 300 pg/ml to 415.3±4.5 pg/ml, increasing from 485±16.9pg/ml to 914.2±9.5 pg/ml

[0273] IX. Analysis of TNFR1 Ectodomain Sheddase Regulatory Activity ofARTS-1 Catalytic Mutants

[0274] The predicted ARTS-1 polypeptide contains a peptidase/proteaseconsensus motif found in the aminopeptidase family of gluzincin zincmetalloproteases. It was determined if this peptidase/protease catalyticmotif was necessary for the ability of ARTS-1 to promote the shedding ofthe TNFR1 ectodomain. To conduct this experiment, a series of mutantscontaining point mutations predicted to abolish the ARTS-1peptidase/protease activity were constructed. The construction of celllines expressing these mutants was conducted as described in the sectionabove, with experimental details provided in Examples 10 and 11. Resultsof this experiment are depicted in FIG. 10.

[0275] The experiment was done in two phases. The first phase involvedconstruction of stably transfected cell lines which expressed eitherwild-type or mutant forms of the ARTS-1 polypeptide. The ARTS-1 mutantsconstructed for this experiment were designed to eliminatemetalloprotease catalytic activity by disrupting the zincmetalloprotease consensus catalytic motif HEXXH(Y)₁₈E (SEQ ID NO:10;consisting of a zinc binding and catalytic site domains). In the ARTS-1polypeptide, this motif is located at H³⁵³ELAH(Y)₁₈E³⁷⁶ (SEQ ID NO:11).Each of the mutations made lies within this domain. These mutations areH353P, E354V, H353P and E354V in combination, and H357V. These mutationshave been previously shown to abolish zinc binding and/or catalytic(enzymatic) activity in proteins containing the motif (Devault et al.,FEBS Lett., 23154-23158 [1988]; Devault et al., J. Biol. Chem.,263:4033-4040 [1988]; Vallee and Auld, FEBS Lett., 257:138-140 [1989];Vallee and Auld, Biochemistry 29:5647-5659 [1990]; and Wang and Cooper,Proc. Natl. Acad. Sci. USA 90:1222-1226 [1993]).

[0276] Six cell lines were created by stably transfecting the NCI-H292cells with the six constructs, all based on the pTarget expressionvector. These constructs (and resulting cell lines) contained:

[0277] 1) an empty pTarget vector,

[0278] 2) the ARTS-1 cDNA (WT) coding region,

[0279] 3) the ARTS-1 cDNA encoding a H353P mutation,

[0280] 4) the ARTS-1 cDNA encoding a E354V mutation,

[0281] 5) the ARTS-1 cDNA encoding a H353P and E354V double mutation,and

[0282] 6) the ARTS-1 cDNA encoding a H357V mutation.

[0283] Following the introduction and selection of these constructs inthe host cell lines, the amount of TNFR1 ectodomain shedding occurringin each of the lines was determined by measuring the levels of sTNFR1ectodomain in cell culture supernatants using a commercially availablesandwich-enzyme-linked immunosorbent assay (ELISA) technique (R & DSystems) with a lower limit of detection of 7.8 pg/ml. These results aredepicted in FIG. 10 as the mean of five independent experiments, as wellas the SEM (standard error of the mean). As shown in this Figure, thecell line containing the recombinant ARTS-1 cDNA with no mutations(ARTS-1) showed a significantly elevated level of sTNFR1 in the culturesupernatant compared to cell lines containing no integrated DNA (WT) orcontaining the empty pTarget vector (MOCK). Unexpectedly, each of thecell lines containing mutant forms of the ARTS-1 polypeptide also showedelevated levels of sTNFR1 compared to the control lines (i.e., WT andMOCK lines). This experiment demonstrates an unexpected property of thepresent invention, as the peptidase/protease activity of the ARTS-1polypeptide appears not to be required for its sTNFR1 sheddingregulatory activity.

[0284] X. Analysis of ARTS-1/TNFR1 Interaction In Vivo

[0285] In light of the of the identification of the ARTS-1 gene by theyeast two-hybrid interaction screening, the physical association ofARTS-1 and TNFR1 was verified in vivo in a mammalian cell culture systemusing a co-immunoprecipitation assay.

[0286] Crude membrane fractions from cultured NCI-H292 cells wereisolated and incubated with murine anti-human TNFR1 monoclonal antibody(R & D System) or 1 ml of anti-ARTS-1 antiserum overnight. Following theincubation, the resulting antibody complexes were immunoprecipitatedusing immobilized protein A/G beads (Pierce), and the precipitatedproteins analyzed by Western immunoblotting.

[0287] Two different combinations of precipitation and immunoblottingantibody were used. The results of these immunoprecipitation experimentsare shown in FIG. 12. In one experiment (FIG. 12, top panel), theanti-TNFR1 antibody was used in the immunoprecipitation (indicated as“IP” in the Figure), and the anti-ARTS-1 antiserum was used as theprimary antibody in the immunoblotting (indicated as “IB” in theFigure). In a second experiment (FIG. 12, bottom panel), the antibodieswere reversed, where the anti-ARTS-1 antiserum was used in theimmunoprecipitation, while the anti-TNFR1 antibody was used as theprimary antibody in the immunoblotting.

[0288] As shown in FIG. 12, immunoprecipitation of the NCI-H292 cellmembrane proteins with an anti-TNFR1 monoclonal antibody resulted in thecoprecipitation of the 100 kDa ARTS-1 species and, conversely,immunoprecipitation with anti-ARTS-1 antiserum coprecipitated the 55 kDaTNFR1. These results indicate an in vivo protein-protein interactionbetween ARTS-1 and TNFR1 proteins.

[0289] Similar immunoprecipitation experiments were also performed usingthe stably-transfected NCI-H292 cell lines described in Example 9. Inthis experiment, the anti-TNFR1 antibody was used to immunoprecipitateprotein from the various membrane protein fractions, and the resultingimmunoprecipitate was examined by Western immunoblotting usinganti-ARTS-1 antiserum as the primary antibody. As shown in FIG. 13,immunoprecipitation using an anti-TNFR1 monoclonal antibody of cellmembrane protein derived from the anti-sense ARTS-1 cell line (AS)showed decreased amounts of ARTS-1 protein as compared tocontrol-transfected (Mock) or non-transfected (WT) cells, consistentwith decreased ARTS-1 protein expression in anti-sense ARTS-1 cells. Noincrease in ARTS-1 protein levels relative to control cell lines wasdetected following immunoprecipitation of ARTS-1 overexpressing celllines with an anti-TNFR1 monoclonal antibody, which likely reflectsincreased TNFR1 shedding related to ARTS-1 over-expression.

[0290] XI. Therapeutic Agents to Treat Immune Diseases and Disorders

[0291] The present invention provides at least one polypeptide whichpromotes the shedding of TNFR1 (i.e., ARTS-1 polypeptide) from thesurface of cultured human cells, a gene encoding the polypeptide,recombinant vectors comprising the gene, host cells comprising thevectors and antibodies directed against the ARTS-1 polypeptide. It iscontemplated that these compositions will find use as therapeutic agentsfor the treatment of TNF-mediated immune diseases. It is contemplatedthat the therapeutic agents or the agents for gene therapy of thepresent invention will be administered to a subject orally,parenterally, systemically or locally. It is also contemplated thatgenes and polypeptides which are substantially homologous to the ARTS-1gene provided by the present invention will also find use in thetreatment of TNF mediated diseases and disorders.

[0292] When compositions of the present invention are used astherapeutic agents or agents for gene therapy for immune diseases, it isnot intended that the present invention be limited to a particulardisease. For example, the gene or polypeptide of the invention may beused, alone or in combination, to treat inflammatory diseases including,but not limited to, rheumatoid arthritis, inflammatory bowel disease,septic shock, cachexia, autoimmune disorders, graft-versus-host diseaseand insulin resistance.

[0293] In some preferred embodiments of the present invention, when thetherapeutic agent of the invention is administered orally, the agent maybe formulated into a tablet, capsule, granule, powder, pill, troche,liquid drops, suspension, emulsion, syrup or the like. Alternatively,the therapeutic agent may be prepared into a dry product which isre-dissolved just before use. In preferred embodiments, when thetherapeutic agent of the invention is administered parenterally, theagent may be formulated for intravenous injection, intramuscularinjection, intraperitoneal injection, subcutaneous injection, as asuppository, etc. Injections are supplied in the form of unit dosageampules or multi-dosage containers. The formulations of the presentinvention may be prepared by conventional methods using appropriateexcipients, fillers, binders, wetting agents, disintegrating agents,lubricating agents, surfactants, dispersants, buffers, preservatives,dissolution aids, antiseptics, flavoring/perfuming agents, analgesics,stabilizers, isotonicity inducing agents, etc. conventionally used inpharmaceutical preparations.

[0294] Each of the above-described formulations may containpharmaceutically acceptable carriers or additives. Specific examples ofsuch carriers or additives include water, pharmaceutically acceptableorganic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone,carboxyvinyl polymers, sodium alginate, water-soluble dextran, sodiumcarboxymethyl amylose, pectin, xanthan gum, gum arabic, casein, gelatin,agar, glycerol, propylene glycol, polyethylene glycol, vaseline,paraffin, stearyl alcohol, stearic acid, serum albumin, mannitol,sorbitol and lactose. One or a plurality of these additives are selectedor combined appropriately depending on the form of the preparation.

[0295] The dosage levels of the therapeutic agent of the invention willvary depending on the age of the subject, the route of administrationand the frequency and duration of administration and may be varied overa wide range as suitable for each subject. When an effective amount ofthe polypeptide or antibody of the invention is administered incombination with an appropriate diluent and a pharmaceuticallyacceptable carrier, the effective amount of the polypeptide or antibodyis in the range from 0.01 to 1000 mg/kg per administration, althoughother amounts are contemplated, as appropriate. One skilled in the artis capable of determining the therapeutically effective amountappropriate any given circumstances. In some embodiments, thetherapeutic agent is administered once a day or in several dosages perday for at least one day.

[0296] In some embodiments of the present invention, at least one geneof the present invention is used as an agent for therapy for immunediseases or disorders. When used as a therapeutic agent, the gene(s) maybe administered systemically or locally. The gene(s) may be delivered bydirect application of the nucleic acid to cells or tissues.

[0297] In one embodiment, the present invention is used as a genetherapy agent to treat an inflammatory disease or condition. In oneembodiment, the gene therapy agent of the present invention is deliveredvia a viral delivery system. In an alternative embodiment, the genetherapy agent of the present invention involves a non-viral deliverysystem.

[0298] Viral-mediated gene delivery has been shown to be an effectivemechanism for gene delivery for use in gene therapy. Indeed, methods forviral-mediated gene therapy have recently been shown to be effective inhuman and non-human systems (Kordower et al., Science 290:767-773[2000]; Lee et al., Nature 408:483-488 [2000]; Cavazzana-Calvo et al.,Science 288:669-672 [2000]; Kay et al., Nature Genetics 24:257-261[2000]; Amado and Chen, Science 285:674-676 [1999]; Burton et al., Proc.Natl. Acad. Sci. USA 96(22):12725-12730 [1999]; Zhang, Cancer GeneTher., 6(2):113-138 [1999]; Connelly et al., Blood 91(9):3273-3281[1998]; and Connelly et al., Blood 88(10):3846-3853 [1996]). A number ofviruses have been demonstrated to be effective or potentially effectivetools in recombinant gene delivery to subjects, including adenovirus(lentivirus) vectors, adeno-associated virus vectors, herpes virusvectors, vaccinia virus vectors, and retrovirus vectors. In somepreferred embodiments, the recombinant viral vector comprising theARTS-1 gene of the present invention comprises nucleic acid elementsoperably linked for the purpose of transcribing and translating the geneof the invention in cells in a subject. In preferred embodiments, thesenucleic acid elements consist of a nucleotide sequence encoding theARTS-1 polypeptide, and operably linked promoter and enhancer elementsfor expression of the ARTS-1 gene. In some embodiments, thesepromoter/enhancer elements are widely active in all or many cell types,and direct constitutive expression of the gene (e.g., cytomegalovirus(CMV), SV40 or Rous sarcoma virus (RSV) promoter/enhancer sequences). Inalternative embodiments, operably linked promoter/enhancer elements arerestricted in activity to a single cell type or tissue (e.g.,cardiac-specific or liver-specific promoter/enhancers) (Maniatis et al.,Science 236:1237-1245 [1987]; Voss et al., Trends Biochem. Sci., 11:287[1986]). In further embodiments, a promoter/enhancer element thatimparts inducible (i.e., conditional) expression of an operably linkedopen reading frame (e.g., tetracycline inducible or repressiblepromoters) is used. Furthermore, in other embodiments, operably linkednucleotide sequences include sequences directing proper translationinitiation, post-transcriptional splicing/editing, and/orpolyadenylation. In still other embodiments, in addition to containingnucleotide sequences controlling the expression of the ARTS-1 gene, aviral gene therapy vector further contains the necessary nucleotidesequences for in vitro replication and propagation of the virus,production of infective virion particles, and sequences that impartstability of the DNA in a cellular host (although many viral functionsrequire the presence of a “helper virus”). Collectively, such sequencesare sometimes referred to as the viral “backbone.”

[0299] Additionally, a genetic sequences of the invention may beenclosed in phospholipid vesicles such as liposomes, and the resultantliposomes administered to a subject. Liposomes are biodegradablevesicles containing an internal aqueous region surrounded by a lipidbilayer. This structure is able to encapsulate materials (e.g., at leastone gene of the present invention). By mixing at least one gene of thepresent invention with phospholipid starting material, a liposome-genecomplex will form. Subsequently, when this complex is cultured withcells, the gene(s) in the complex is taken into the cells (i.e., vialipofection). A liposome-gene complex comprising at least one gene ofthe present invention can be administered to a subject, either locallyor systemically. In addition to liposomes, a plasmid encoding thegene(s) of interest may be used.

[0300] Alternatively, direct DNA administration, liposome gene transfer,or viral vector, all comprising at least one gene of the presentinvention, can be used to transfect cells ex vivo (i.e., not within thesubject), followed by the transplantation of the recipient cells intothe subject. The source of the cells receiving the gene(s) of theinvention can be cells that have been removed from the subject, or cellsfrom some other source. Following delivery of the gene(s) of the presentinvention into these cells, the cells are then placed into the subjectto provide therapeutic value.

[0301] In some embodiments of the present invention, methods arecontemplated for administering gene(s) of the invention locally totissues via surgical or injection protocols, as well as systemically,such as by intravenous or intra-arterial administration. Further, anadministration method combined with catheter techniques and surgicaloperations may also be employed.

[0302] The dosage levels of the agent for delivering the gene(s) of theinvention vary depending on the age, sex and conditions of the subject,the route of administration, the number of times of administration, andthe type of the formulation, among other considerations. One skilled inthe art is capable of determining the therapeutically effective amountappropriate any given circumstances. Usually, it is appropriate toadminister a gene of the invention in an amount of 0.1-100 mg/adultbody/day, although other concentrations are contemplated, asappropriate.

[0303] XII. Diagnostic Agents for Immune Diseases and Disorders

[0304] It is contemplated that the ARTS-1 gene of the present inventionwill find use as a diagnostic marker for TNF signaling activity. Indeed,levels of ARTS-1 mRNA or ARTS-1 polypeptide in biological samples (e.g.,blood, urine, serum or any other body fluid) are usefuil indicators ofthe levels of TNF signaling activity in vivo. It is contemplated thatthe presence of elevated ARTS-1 mRNA and/or polypeptide levels correlatewith decreased TNF signaling activity, while reduced levels of ARTS-1mRNA or polypeptide correlate with increased TNF activity. It iscontemplated that excessive or inadequate TNF activity is indicative ofdisease or pathological states. Thus, the present invention providesmethods and compositions for rapid quantitation of ARTS-1 mRNA andpolypeptide indicative of TNF signaling activity, thereby providinguseful tools for assessing the immune condition of an individualsuspected of suffering from TNF-mediated immune disorders or diseases.In contrast, existing methods for the assay of TNF activity involvelengthy tissue culture assays which are not readily applicable for useas a rapid diagnostic tool in a clinical setting (Suffredini et al., J.Immunol., 155:5038-5045 [1995]; Suffredini et al., N. Eng. J. Med.,321:280-287 [1989]; and Eskandari et al., Immunol. Invest., 19:69-79[1990]).

[0305] The present invention further provides compositions for use indiagnostic kits which can be used to rapidly assess ARTS-1 mRNA orpolypeptide using either a nucleic acid probe specific for ARTS-1 mRNA,or PCR primers capable of amplifying ARTS-1 mRNA (all derived from thenucleic acid of SEQ ID NO:1) or the antibody directed against at least aportion of an ARTS-1 polypeptide. Such kits may be designed toincorporate PCR, nucleic acid probe hybridization, and/or antibodyimmunoassay protocols for ARTS-1 marker detection. These kits mayfurther include any reagent(s) or material(s) which makes possible orfacilitates the analysis of a sample (e.g., apparatus for samplecollection, sample tubes, holders, trays, racks, dishes, plates,instructions to the kit user, solutions or other chemical reagents, andsamples to be used for standardization, normalization, and or as controlsamples).

[0306] XIII. Identification of Genes Substantially Homologous to ARTS-1

[0307] In other embodiments, the present invention provides compositionsand methods for the identification of genes substantially homologous tothe ARTS-1 gene (i.e., SEQ ID NO:1). It is contemplated that genessimilar to the gene set forth in SEQ ID NO:1 have the ability toregulate the cleavage and shedding of TNFR1 ectodomain. In particular,it is contemplated that the compositions and methods of the presentinvention will find use in stringent hybridization and/or PCR methods toidentify genes substantially homologous to SEQ ID NO:1.

[0308] It is further contemplated that genes similar to the gene setforth in SEQ ID NO:1 have the ability to regulate the cleavage andshedding of the ectodomains of other pro-inflammatory cytokine receptors(e.g., type II IL-1 receptor and IL-6 receptor). As discussed above,TNF, IL-1 and IL-6 all are multi-functional pro-inflammatory cytokineswhich regulate acute phase protein production during innate immuneresponses to infection and tissue injury (Suffredini et al., J. Clin.Immunol., 19:203-214 [1999]). Consequently, it is contemplated thatgenes significantly homologous to ARTS-1 will find utility in thetreatment of immune disorders or diseases mediated by abnormal TNF, IL-1or IL-6 activity. It is contemplated that the methods and compositionsof the present invention will find use in promoting the cleavage andshedding of the TNFR1 ectodomain, as well as the ectodomains of IL-1 andIL-6 cytokine receptors. Although it is not intended that the presentinvention be so limited, two methods for isolation of genessubstantially homologous to the ARTS-1 gene are provided below.

[0309] A. Hybridization to Identify Genes Significantly Homologous tothe ARTS-1 Gene

[0310] In this method, a probe derived from the nucleic acid of SEQ IDNO:1 is used to screen a phage EDNA library. The probe is preferablyderived from the ARTS-1 gene coding region. The probe may be anoligonucleotide amplified or excised from a larger nucleic acid (e.g.,from a purified restriction digest product of a plasmid or other vector)or produced synthetically, recombinantly or by PCR amplification. Thereis no limitation on the size of the probe, although it is preferablylonger than 25 nucleotides, and most preferably encompasses the entirecoding region of the ARTS-1 cDNA. This probe may be single or doublestranded. In particularly preferred embodiments the probe is labelledwith a “reporter molecule,” so that is detectable in a detection systemof choice. A detection system may include, but is not limited to,enzymatic detection, fluorescence, radioactivity, and luminescentsystems. Indeed, it is not intended that the present invention belimited to any particular detection system or label.

[0311] As a source of nucleic acid to be used in the hybridizationscreening, it is contemplated that nucleic acid from a wide variety ofeukaryotic sources may be used. However, λgt10-based EDNA bacteriophagelibraries derived from human sources are preferred and advantageous inthis embodiment of the invention. Nonetheless, the source of the nucleicacid to be screened is by no means limited.

[0312] Example 1 provides experimental protocols used in the developmentof the present invention, and specifically, a protocol for the screeningof a phage plaque library with a radiolabelled DNA probe. This sameprotocol finds use in identification of genes which are substantiallyhomologous to SEQ ID NO:1. Briefly, a bacteriophage cDNA library isplated in a lawn of DH-5α host bacteria. The DNA contained in each ofthe plaques is replica plated via a plaque-lift onto a membrane suitablefor subsequent hybridization. The DNA is fixed to the filter, and probedin solution using the ARTS-1 derived probe under stringent conditions.Phage plaque DNA with the ability to hybridize to the probe is isolated,analyzed and sequenced.

[0313] B. PCR to Identify Genes Significantly Homologous to the ARTS-1Gene

[0314] PCR may be used to identify genes significantly homologous to theARTS-1 gene. In a preferred embodiment, a sense primer and an anti-senseprimer derived from the polypeptide coding region of the ARTS-1 gene aresynthesized and used in a polymerase chain reaction (PCR). In analternative embodiment, “degenerate” PCR primers are used. In theseembodiments, the PCR primers have nucleotide sequences which encodeamino acid domains of the ARTS-1 polypeptide, but differ from the ARTS-1gene nucleotide sequence. Criteria for designing “degenerate” PCRprimers are well known in the art. It is not intended that the presentinvention be limited to any particular primer or primer set.

[0315] It is also contemplated that nucleic acid from a wide variety ofeukaryotic sources may be used as template in the PCR methods. It is notintended that the present invention be limited as to the source of thetemplate nucleic acid. However, nucleic acid template derived from humansources is the most preferred embodiment of the present invention.

[0316] In preferred PCR methods, a nucleic acid that is substantiallyhomologous to the ARTS-1 gene is amplified using materials and methodsknown in the art. Conditions of the PCR may be manipulated (e.g., theduration or temperature of the cycle times may be changed) to amplify anucleic acid substantially homologous to the ARTS-1 gene. Indeed, it iscontemplated that many suitable PCR conditions as known in the art willfind use in the present invention. The nucleic acid amplified in the PCRreaction is visualized, isolated, subeloned, and sequenced usingtechniques standard in the art.

[0317] The isolated nucleic acid obtained by either plaque hybridizationor PCR is further examined to determine if the isolated nucleic acid is“substantially homologous” to the ARTS-1 gene of the present inventionbased on criteria previously discussed. In a preferred embodiment, theisolated nucleic acid which is substantially homologous to the ARTS-1gene encodes a polypeptide having the ability to promote the cleavageand shedding of at least one of the ectodomains of the group of cytokinereceptors consisting of TNFR1, and IL-1 and IL-6 cytokine receptors. Thenucleic acid obtained is then tested using methods such as thosedescribed in Examples 7, 11, 12 and 13. In a most preferred embodiment,the isolated gene has TNF regulatory activity, as determined using theprotocol supplied in Example 14.

[0318] XIV. Methods for Drug Screening

[0319] It is contemplated that compounds which are able to regulateARTS-1 activity or receptor ectodomain shedding are candidates forfurther development as therapeutically advantageous drugs. Such drugsfind use as immune response modifiers, to either enhance or attenuatethe action of an cytokine signalling (e.g., TNF, IL-1 or IL-6). Thepresent invention provides compositions and methods for the screeningand identification of test compounds which can regulate ARTS-1 activity,ARTS-1 transcript or protein expression, and receptor shedding, andthus, identifies drug candidates for further development astherapeutics. However, an understanding of the mechanism(s) of how aparticular compound regulates cytokine signalling and proinflammatoryimmune responses is not necessary in order to use the present invention.

[0320] A. Method for Drug Screening to Identify Compounds Having theAbility to Regulate ARTS-1 Expression

[0321] The present invention provides screening methods to identifycompounds which can regulate the level of ARTS-1 transcript or proteinin a tissue. Test compounds which are able to regulate ARTS-1 transcriptor protein levels in a cell or tissue are candidates for furtherdevelopment as therapeutic agents. It is contemplated that compoundswhich can upregulate or downregulate ARTS-1 expression also have theability to downregulate or upregulate cytokine signalling (i.e., aproinflammatory immune response), respectively.

[0322] In one embodiment of the present invention, the screening methoduses Northern blotting to assess the levels of the ARTS-1 transcript ina cell culture following exposure of the culture to a test compound. Inthis embodiment, cultured cells are exposed to a test compound, andsamples of tissue are collected at intervals ranging from approximately0 to 48 hours. RNA is isolated from these cells and subjected toNorthern blot analysis, as described in Example 3, using a probespecific for the ARTS-1 mRNA. Comparison of the ARTS-1 transcript levelsbefore and after exposure to the test compound identifies thosecompounds that upregulate or downregulate ARTS-1 transcript levels. Itis contemplated that compounds that can regulate ARTS-1 transcriptlevels provide targets for further development as therapeutic agents.

[0323] In a preferred embodiment, cultured human NCI-H292 pulmonarymucoepidermoid carcinoma cells are used in the screening. However, it isnot intended that the invention be limited to the use of only this celltype, as other cell types are equally suitable, and are known to thosein the art. Similarly, it is not intended that the reagents, methods orapparatus for RNA collection and analysis be limited to those describedherein, as numerous suitable equivalents are known to those in the art.

[0324] In another embodiment of the screening method of the presentinvention, Western immunoblotting is used to assess the levels of ARTS-1protein following exposure of a cell culture to a test compound. In thisembodiment, cultured cells were exposed to a test compound, in thiscase, 4b-phorbol 12-myristate 13-acetate (PMA), and samples of tissuewere collected at 0, 2, 8 and 24 hours following the exposure. Membraneproteins were isolated from these cells and subjected to Westernimmunoblot analysis, as described in Examples 4 and 5, using antiserumspecific for the ARTS-1 protein. Results of this screening are shown inFIG. 14, Panel A. Comparison of the ARTS-1 protein levels in the cellsbefore and after exposure to the test compound demonstrated thattreatment of the cells with PMA resulted in an upregulation of ARTS-1protein expression. It is contemplated that compounds that canupregulate ARTS-1 protein levels are therapeutically advantageous, assuch compounds can suppress a proinflammatory immune response byenhancing receptor ectodomain shedding. However, an understanding of themechanism(s) is not required in order to use the present invention. Asindicated herein, PMA is a candidate for further development as atherapeutic agent.

[0325] In an alternative preferred embodiment, cultured human NCI-H292pulmonary mucoepidermoid carcinoma cells are used in the screening.However, it is not intended that the invention be limited to the use ofonly this cell type, as other cell types are equally suitable, and areknown to those in the art. Similarly, it is not intended that thereagents, methods or apparatus for protein collection and Westernimmunoblot analysis be limited to those described herein, as numeroussuitable equivalents are known to those in the art.

[0326] B. Method for Drug Screening to Identify Compounds Having theAbility to Enhance Receptor Ectodomain Shedding

[0327] The present invention provides screening methods to identifycompounds which can regulate receptor ectodomain shedding. Testcompounds which are able to regulate ectodomain shedding are identifiedas candidates for further development as therapeutic agents. It iscontemplated that compounds which can upregulate or downregulateectodomain shedding also have the ability to downregulate or upregulatea proinflanunatory immune response, respectively.

[0328] In one embodiment of the present invention, the screening methoduses an ELISA to assess the levels of sTNFR1 in the supernatant of cellcultures. In the experiments described herein, an ELISA was used toassess the levels of sTNFR1 in the supernatants from human NCI-H292pulmonary mucoepidermoid carcinoma cells following exposure of theculture to PMA. In this embodiment, the cultured cells were exposed tothe compound, and samples of supernatant were collected at 0, 2, 8 and24 hours following the exposure. The samples were analyzed using anELISA with an anti-sTNFR1 antibody as described in Example 11. Theresults of this ELISA screening are shown in FIG. 14, Panel B. As can beseen in the Figure, treatment of the cultured cells resulted in anincrease in sTNFR1 shedding over the course of 24 hours compared to acontrol culture (n=5,*P<0.05). Thus, this screening method identified acompound as a candidate for further drug development.

[0329] It is contemplated that compounds that can upregulate sTNFR1ectodomain shedding are therapeutically advantageous, as such compoundscan suppress a proinflammatory immune response. However, anunderstanding of the mechanism(s) is not required in order to use theinvention. Based on the criteria set forth and described herein, PMA isa candidate for further development as a therapeutic agent.

[0330] It is not intended that the method for drug screening assessingsTNFR1 ectodomain shedding be limited to analysis of sTNFR1 shedding. Itis contemplated that the analysis of ectodomain shedding other cytokinereceptors, such as type II interleukin-1 cytokine receptor andinterleukin-6 cytokine receptor alpha-chain gp80 also find use with thepresent invention.

[0331] C. Method for Drug Screening to Identify Compounds Having theAbility to Regulate the Peptidase Activity of ARTS-1 Protein

[0332] The present invention provides screening methods to identifycompounds which can regulate the peptidase activity of ARTS-1 protein.Test compounds which are able to regulate ARTS-1 peptidase activity arecandidates for further development as therapeutic agents. It iscontemplated that compounds which can upregulate or downregulate ARTS-1peptidase activity also have the ability to downregulate or upregulate aproinflammatory immune response, respectively.

[0333] In one embodiment of the present invention, the screening methoduses amino acid p-nitroanilide substrates to assess the aminopeptidaseactivity (described in Example 7) of purified recombinant GST-ARTS-1fusion protein (described in Example 6). In this screening method, theamino acid p-nitroanilide hydrolysis reaction is conducted in theabsence and presence of a test compound, according to the reactionconditions provided in Example 7, and the rate of amide bond hydrolysisby ARTS-1 is determined. It is then observed whether a test compound hasthe ability to regulate the rate of amide bond hydrolysis.

[0334] It is contemplated that compounds that can regulate ARTS-1 amidebond hydrolysis are therapeutically advantageous, as such compounds canregulate a proinflammatory immune response, and are targets for furtherdevelopment as therapeutic agents. However, an understanding of themechanism(s) is not required in order to use the present invention.

[0335] It is not intended that this method for drug screening be limitedto those reagents itemized in Example 7. For example, it is contemplatedthat purified ARTS-1 proteins in addition to ARTS-1-GST also find usewith the present invention. Furthermore, it is contemplated that morethan one amino acid p-nitroanilide substrate finds use with thescreening method, as isoleucine p-nitroanilide, phenylalaninep-nitroanilide and glycine p-nitroanilide substrates can all be used inthe hydrolysis reaction.

EXPERIMENTAL

[0336] In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); Accurate Chemical and Scientific Corporation(Accurate Chemical and Scientific Corporation, Westbury, N.Y.); AdvancedBiotechnologies (Advanced Biotechnologies Incorporated, Columbia, Md.);Amersham/Pharmacia (Amersham/Pharmacia Biotech, Piscataway, N.J.); ATCC(American Type Culture Collection, Rockville, Md.); Bachem (Bachem, Kingof Prussia, Pa.); Biofluids (Biofluids, Inc, Rockville, Md.); BoehringerMannheim (Roche/Boehringer Mannheim Corporation, Indianapolis, Ind.);Calbiochem (Calbiochem-Novabiochem Corp, San Diego, Calif.); Clontech(Clontech Laboratories, Inc., Palo Alto, Calif.); Genzyme (GenzymeCorporation, Cambridge, Mass.); Life Technologies (LifeTechnologies/Gibco/BRL, Gaithersburg, Md.); Novex (Novex/Invitrogen,Carlsbad, Calif.); Pierce (Pierce, Rockford, Ill.); Promega (PromegaCorporation, Madison, Wis.); R & D Systems (R & D Systems, Minneapolis,Minn.); Research Genetics (Research Genetics, Huntsville, Ala.); Roche(Roche/Boehringer Mannheim Corporation, Indianapolis, Ind.); Sigma(Sigma Chemical Co., St. Louis, Mo.); Stratagene (Stratagene, La Jolla,Calif.).

[0337] The following Examples are provided in order to demonstrate andfurther illustrate certain preferred embodiments and aspects of thepresent invention and are not to be construed as limiting the scopethereof

Example 1 Identification of ARTS-1, a Novel Human AminopeptidaseRegulator of Type1 Tumor Necrosis Factor Receptor (TNFR1) Shedding

[0338] A) Yeast Two-Hybrid Library Screening

[0339] A yeast two-hybrid library screening was conducted to identifyproteins that interact with or are capable of interacting with theextracellular domain of the type I, 55 kDa TNF receptor (TNFR1). A humanlung cDNA library (Clontech), cloned into pGAD10 (GAL4 DNA-activationdomain vector), was screened to detect interactions with a GAL4BD-TNFR1fusion protein using a Matchmaker System 2 (Clontech) using methodsknown in the art as well as the manufacturer's recommended protocols.Amino acids 26 to 216 of TNFR1, corresponding to the extracellulardomain (i.e., the ectodomain) located between the putative leader domainand the transmembrane domain (Nophar et al., EMBO J., 10:3269-3278[1990]) were cloned into pAS2-1 (GAL4 DNA-binding domain vector) togenerate the bait GAL4 DNA-binding domain fusion protein, GAL4BD-TNFR1.The Y190 yeast strain was transformed with the pAS2-1 GAL4BD-TNFR1 baitplasmid by the lithium acetate method utilizing the YeastmakerTransformation System (Clontech). Transformed yeast were selected onsynthetic drop-plates deficient in tryptophan, leucine and histidine inthe presence of 25 mM 3 amino-1,2,4-triazole (SIGMA). His⁺ colonies werethen subjected to β-galactosidase colony-lift filter assays andβ-galactosidase producing colonies were selected and restreaked onsynthetic drop-out plates. Approximately 7.11×10⁶ transformants werescreened. β-galactosidase positive colonies which produced a blue signalwithin 2 hours were selected for further study. Selected colonies wereanalyzed for expression of the GAL4 binding domain (GAL4BD) fusionprotein by immunoblotting using an anti-GAL4BD monoclonal antibody(Clontech). Thirty three positive clones were identified which activatedthe β-galactosidase reporter construct. All 33 positive clones weresequenced using an ABI Perkin Elmer 377 automated fluorescent sequencer.

[0340] One positive clone, L26C-53A, encoding a consensus zincmetalloprotease catalytic motif was selected for further study. Thisclone contained a 2355-bp insert containing an open reading frame of 631amino acids, but did not contain a poly(A) tail or a putativetranslation initiation site. The gene encoded by this insert wasentitled aminopeptidase regulator of type-1, 55 kDa tumor necrosisfactor receptor shedding (or ARTS-1). The L26C-53A sequence correspondsto ARTS-1 bases 1044 to 3082.

[0341] B) ARTS-1 cDNA Cloning/Phage Plaque Hybridization

[0342] Using ³²P-labelled DNA derived from clone L26C-53A as a probe, aPMA-stimulated NCI-H292 cell line cDNA library (Stratagene) was screenedto obtain ARTS-1 cDNA clones. The NCI-H292 cell line was derived fromhuman pulmonary mucoepidermoid carcinoma cells and has been demonstratedto shed cell surface sTNFR1 in response to PMA stimulation (Levine etal., Am. J. Respir. Cell Mol. Biol., 14:254-261 [1996]). The uni-ZAP XRphage cDNA library (Stratagene) was constructed with NCI-H292 cell poly(A⁺) mRNA isolated following 24 hours of stimulation with 1 μM PMA(phorbol 12-myristate, 13-acetate, Sigma).

[0343] Bacteriophage from the uni-ZAP library were plated in a lawn ofXL1-Blue (MRF-) E. coli (Stratagene) at a density of 50,000 pfu per 150mm plate and incubated overnight at 37° C. Plaques were transferred toHybond N+ filters (Amersham/Pharmacia) and denatured for 5 minutes in1.5M sodium chloride, 0.5 M sodium hydroxide. Filters were thenneutralized in two washes of 5 minutes each in 1.5M sodium chloride, 1MTris-base, rinsed in 3×SSC and UW cross-linked. Filters werepre-hybridized in 10×Denhardt's solution, 6×SSPE, 1% SDS at 42° C. for 4hours in hybridization buffer. Filters were hybridized overnight at 42°C. with approximately 10⁷ CPM of ³²P-labelled L26C-53A insert which wasgenerated by random priming. Filters were then washed in 2×SSC, 0.5% SDSfor 20 minutes at room temperature, followed by two 1 hour washes in1×SSC, 0.1% SDS at 65° C. Filters were then exposed to x-ray filmovernight and positive plaques were selected. Positive plaques weresubjected to two additional rounds of plaque hybridization prior tosequencing. Positive plaques were recovered via in vivo excisionutilizing the ExAssist helper phage (Stratagene).

[0344] Following 3 rounds of screening, four hybridizing phage cloneswere identified, then sequenced utilizing the ThermoSequenase cyclesequencing kit (Amersham/Pharmacia). These four hybridizing clonesoverlapped the L26C-53A sequence, but none encoded a full-length cDNA.Sequencing revealed one phage clone (bp 1-1777) which contained theputative 5′ UTR and three phage clones which contained the putative 3′UTR and the poly(A) tail (bp 2181-4845). cDNA sequence encoding theportion of the gene lying between these 5′ and 3′ terminal clones wasobtained via PCR from Marathon-Ready™ human lung cDNA (Clontech)utilizing Pfu Turbo DNA Polymerase (Stratagene) and the followingprimers: (SEQ ID NO:3) 5′-ATAACCATCACAGTGAGGGGGAGG-3′ and (1684-2279)(SEQ ID NO:4) 5′-TAGTTGACTCCGCAGCATTCGCTC-3′ (2257-2280)

[0345] The cDNA segment amplified with these primers was cloned intopGemTEasy (Promega) and both strands were sequenced by automatedfluorescent sequencing using an ABI Perkin Elmer 377 automatedfluorescent sequencer. Sequences of all overlapping regions from theoriginal two-hybrid clone L26C-53A (bp 1044 to 3082), the four NCI-H292cDNA library clones and the PCR product (bp 1684 to 2280) showed nodiscrepancies in the nucleotide sequence in their overlapping regions.

Example 2 Molecular Analysis of the ARTS-1 cDNA and PredictedPolypeptide

[0346] The complete cDNA corresponding to the human ARTS-1 transcriptionunit contains 4845 nucleotides and encodes an open reading frame of 2823bp, as shown in FIG. 1 and set forth in SEQ ID NO:1. The first in-frameATG codon, located at nucleotide 88, follows an in-frame stop codon,located at nucleotide 76, and matches the −3 and +4 nucleotides of theconsensus Kozak sequence consistent with a strong initiator codon(aagatgg) (Kozak, J. Cell Biol., 108:229-241 [1989]). Sequence analysisrevealed five potential N-glyeosylation sites and a TAA stop codon,located at nucleotide 2911. The putative 3′ untranslated regioncontained a consensus polyadenylation site (AATAAA), located atnucleotides 4876 to 4800, which is 18 nucleotides upstream of a 27nucleotide poly(A) tail. The 3′ untranslated region also included twoATTTA sites, located at nucleotides 3929 and 4457; this sites have beenidentified as an mRNA destabilization motif in mRNA's for cytokines,oncogenes and transcriptional activating factors (Stephens et al., J.Biol. Chem., 267:8336-8341 [1992]).

[0347] The open reading frame predicted from the human ARTS-1 cDNAencodes a protein of 941 amino acid residues (See, FIG. 1 and SEQ IDNO:2) with a calculated molecular weight of 107,227 Da and an estimatedpI of 6.0. Sequence analysis, including Kyte-Doolittle hydropathyprediction (Kyte and Doolittle, J. Mol. Biol., 157:105-132 [1982]) wasperformed using MacVector 7.0 software (Oxford Molecular). The locationof the putative hydrophobic transmembrane α-helical domain was predictedutilizing several web-based analysis programs (MEMSAT2 (McGuffin et al.,Bioinform., 16:404-405 [2000]; Sosui (Hirokawa et al., Bioinform.,14:378-379 [1998]; TMAP (Persson and Argos, J. Mol. Biol., 237:182-192[1994]); TMpred (Hofmann and Stoffel, Biol. Chem. Hoppe-Seyler 374:166[1993]; and TopPred2 (von Heijne, J. Mol. Biol., 225:487-494 [1992]). Insum, ARTS-1 is predicted to be a type II integral membrane protein witha single hydrophobic transmembrane α-helical domain, located betweenamino acids 5 and 28 (See, FIGS. 1 and 3), and a very short hydrophobicintracellular amino-terminal domain (See, McGuffin et al., Bioinform.,16:404-405 [2000]; Hirokawa et al., Bioinform., 14:378-379 [1998];Persson and Argos, J. Mol. Biol., 237:182-192 [1994]); Hofmann andStoffel, Biol. Chem. Hoppe-Seyler 374:166 [1993]; and von Heijne, J.Mol. Biol., 225:487-494 [1992]).

[0348] The protein sequence analysis using MacVector software revealed azinc metalloprotease consensus catalytic motif (HEXXH(Y)₁₈E) (SEQ IDNO:10), which is predictive of an active metalloprotease (Hooper, ZincMetalloproteases in Health and Disease, Taylor & Francis, London,England [1996], p.1-21). Utilizing various web-based analysis programs,a putative amino acid transmembrane domain (as predicted by von Heijne,J. Mol. Biol., 20:487-494 [1992]), extending from amino acids 5 to 28,was identified. These features suggest that human ARTS-1 is a type IIintegral membrane protein with a single hydrophobic transmembraneα-helical domain, located between amino acids 5 and 28 (See, FIGS. 1 and2), and a very short, hydrophobic intracellular amino-terminal domain(See, McGuffin et al., Bioinform., 16:404-405 [2000]; Hirokawa et al.,Bioinform., 14:378-379 [1998]; Persson and Argos, J. Mol. Biol.,237:182-192 [1994]); Hofinann and Stoffel, Biol. Chem. Hoppe-Seyler374:166 [1993]; and von Heijne, J. Mol. Biol., 225:487-494 [1992]).

[0349] A BLAST protein homology comparison revealed that the humanARTS-1 contained significant homology with several members of theaminopeptidase family of gluzincin zinc metalloproteases (See, Tables 1and 2, supra) (Wang and Cooper, Zinc Metalloproteases in Health andDisease, Taylor & Francis, London, England [1996]). As has previouslybeen reported for other aminopeptidase family members, the human ARTS-1was found to contain a 375 amino acid domain that is highly conservedwith human placental leucine aminopeptidase (PLAP) (Rogi et al., J.Biol. Chem., 271:56-61 [1996]), rat insulin-regulated aminopeptidase(IRAP) (Keller et al., J. Biol. Chem., 270:23612-23618 [1995]), humanaminopeptidase A (AMP A) (Nanus et al., Proc. Natl. Acad. Sci. USA90:7069-7073 [1993]; Li et al., Genomics 17:657-664 [1993]), humanaminopeptidase N (AMP N) (Olsen et al., FEBS Lett., 238:307-314 [1988]),human puromycin sensitive aminopeptidase (PSA) (Tobler et al., J.Neurochem., 68:889-897 [1997]), rat thyrotropin-releasing hormonedegrading enzyme (TRH DE) (Schauder et al., Proc. Natl. Acad. Sci. USA91:9534-9538 [1994]), Saccharomyces cerevisiae aminopeptidase YSCII(Garcia-Alvarez et al., Eur. J. Biochem., 202:993-1002 [1991]), C.elegans cosmid F49E8.3 gene product (Wilson et al., Nature 368:32-38[1994]), and Lactococcus lactis aminopeptidase N (Tan et al., FEBSLett., 306:9-16 [1992]). This highly conserved domain contains theputative consensus zinc binding domain and catalytic site(T³⁵⁰VAHELAHQWFG (SEQ ID NO:8) and L³⁷²WLNEGFA (SEQ ID NO:9) which ischaracteristic of aminopeptidase family members (Wang and Cooper, ZincMetalloproteases in Health and Disease, Taylor & Francis, London,England [1996]; Keller et al., J. Biol. Chem., 270:23612-23618 [1995]).Furthermore, PLAP, AMP A, AMP N, IRAP and TRH DE share structuralsimilarity with human ARTS-1, based upon the presence of a transmembranedomain and a short intracytoplasmic tail, consistent with a type IIintegral membrane protein.

Example 3 ARTS-1 mRNA Expression Analysis

[0350] Following analysis of the ARTS-1 cDNA, the expression pattern ofendogenous of ARTS-1 mRNA was investigated using a Northernimmunoblotting protocol. RT-PCR was performed on human lung poly(A⁺)mRNA (Clontech) to generate the human ARTS-1 cDNA coding sequenceutilizing the following primers: (SEQ ID NO:5)5′-GCAAGAAGATGGTGTTTCTGCCCCTC-3′ and   (80-105) (SEQ ID NO:6)5′-TTACATACGTTCAAGCTTTTCACT-3′. (2890-2913)

[0351] The full length ARTS-1 coding sequence amplified by these primerswas cloned into the pTarget mammalian expression vector (Promega). TheDNA sequence of this cloned open reading frame was obtained from bothstrands by automated fluorescent sequencing, as known in the art. Therewere no discrepancies between the sequence obtained from these twostrands, nor from the sequence obtained from the PMA-stimulated NCI-H292cell line cDNA library. A ³²P-labelled cDNA probe, corresponding to thefull length ARTS-1 coding sequence, was utilized as a probe for Northernblotting of human multiple tissue Northern blots (Clontech) according tothe manufacturer's protocol.

[0352] As shown in FIG. 3, Northern blot analysis utilizing poly(A+)mRNA from multiple human tissues revealed that the human ARTS-1transcript was expressed in multiple tissues, including spleen, thymus,small and large intestine, peripheral blood leukocyte, heart, placenta,lung, skeletal muscle, kidney and pancreas. In these tissues, anapproximately 5.7 kB predominant mRNA species was detected (See, FIG. 3,top panel). Also shown is the same blot following stripping andrehybridization to a probe specific for the human GAPDH transcript as areference for RNA loading normalization (See, FIG. 3, bottom panel).

Example 4 The Generation of Polyclonal Anti-ARTS-1 Antiserum

[0353] In order to conduct studies of the ARTS-1 polypeptide, polyclonalantiserum was generated against the ARTS-1 polypeptide. Specifically, a17 amino acid ARTS-1 synthetic peptide was used to immunogenize NewZealand white rabbits and subsequently collect immune serum using acommercial service (Research Genetics). This peptide corresponded toamino acids 538 to 554 of the ARTS-1 protein and had the sequence:

[0354] R⁵³⁸GRNVHMKQEHYMKGSD⁵⁵⁴ (SEQ ID NO:7)

[0355] This particular peptide was chosen based upon its antigenicpotential and its lack of homology with other protein sequences viaBLAST homology search.

[0356] Rabbits were immunized with this peptide using standardtechniques. The ARTS-1 peptide was conjugated to KLH and mixed with anequal volume of Freund's complete adjuvant. The amount of antigenutilized per immunization was 0.1 mg, which was injected into threesubcutaneous dorsal sites. The animals received boosts at weeks 2, 6,and 8. Bleeds were obtained at weeks 4, 8 and 10 and tested for thepresence of anti-ARTS-1 antibody. In subsequent experiments, theantiserum obtained from the 10 week bleed was used.

Example 5 Analysis of ARTS-1 Polypeptide Expression in Cultured CellLines and Primary Cells

[0357] Following the production of ARTS-1 polyclonal antiserum, theexpression of endogenous ARTS-1 polypeptide in cultured cell lines andprimary cells was investigated using standard Western immunoblotting asknown in the art. Protein concentrations were determined via the BCAprotein assay (Pierce). Following protein quantitation, 20 microgramsamples of protein were boiled for 5 minutes in Laemmli buffer. Sampleswere then resolved via SDS-PAGE (6% polyacrylamide) and electroblottedonto nitrocellulose (Novex). Blots were incubated overnight in blockingbuffer (5% wt/vol nonfat dry milk in PBS/0.1% Tween-20), then incubatedfor 2 hours with ARTS-1 antiserum at a 1:20,000 dilution in blockingbuffer. Membranes were washed three times for 5 minutes each wash inPBS/0.1% Tween; incubated for 2 hours with 0.8 mg/ml horseradishperoxidase conjugated goat anti-rabbit IgG (Life Technologies) dilutedto 1:5,000 in blocking buffer, then washed three times for 5 minuteseach wash in PBS/0.1% Tween and finally washed three times for 5 minuteseach wash in PBS/0.3% Tween. Membranes were then incubated inchemiluminescent detection substrate for 1 minute and signal detected onX-ray film.

[0358] Crude homogenates made from NCI-H292 cells for immunoblotanalysis were produced by cell lysis in homogenization buffer consistingof 200 μl of 50 mM Tris-HCl, pH 7.2, containing 0.1% Triton X-100 andComplete™ protease inhibitor cocktail tablet (Boehringer Mannheim) andsonicated for four times at 15 seconds each using a microprobe. Thehomogenate was centrifuged at 1,000×g for 5 minutes to remove nuclei,unbroken cells and debris. The low speed supernatant was either utilizedas a whole cell lysate or was further fractionated byultracentrifugation at 100,000×g for 1 hour to generate a crudecytosolic and membrane fractions. The crude membrane fraction wasresolubilized in homogenization buffer prior to immunoblotting.

[0359] Specificity of the resulting polyclonal antiserum was firsttested using crude whole cell homogenates, and membrane and cytosolicfractions prepared from cultured NCI-H292 cells. These results are shownin FIG. 4. Comparing Panels A and B of FIG. 4, the antiserum was shownto detect a predominant 100 kDa membrane form and a predominant 68 kDacytosolic form from the NCI-H292 cells. The whole cell extracts revealeda mixture of these two forms. The preimmune serum showed no reactivitytowards the same samples when used at the same concentration.

[0360] Specificity of the immune serum was further demonstrated incompetition experiments as shown in Panels C and D of FIG. 4. In thesecompetition experiments, 1 μl of the ARTS-1 antiserum was pre-incubatedwith 1 mg of either bovine serum albumin or RGRNVHMKQEHYMKGSD peptide(SEQ ID NO:7) for two hours prior to utilization for immunoblotting.Preincubation of the immune serum with the peptide against which thepolyclonal antiserum was raised resulted in almost complete attenuationof the immune signal (Panel D). In contrast, preincubation of the immuneserum with bovine serum albumin resulted in minimal attenuation ofimmune signal (Panel C).

[0361] The expression of endogenous ARTS-1 polypeptide in primary cellsand other cell lines was further investigated using the anti-ARTS-1antiserum and Western immunoblot technique, as shown in FIG. 5. Theseexperiments analyzed membrane and cytosolic fractions made from humanbronchial brushing specimens, the airway epithelial cell lines BEAS-2Band BET-1A, human lung carcinoma cell line A549, cultured NCI-H292cells, primary cultures of normal human bronchial epithelial cells(NHBE), human umbilical vein endothelial cells (HUVEC) and humanfibroblasts. These experiments also revealed multiple sized forms ofARTS-1 polypeptide on the Western immunoblot, including 132, 100 and 68kDa forms which may localize to various subcellular fractions. Thesemultiple sized forms may be due to regulated processing of the ARTS-1polypeptide, and furthermore may indicate that this processing iscompartmentalized.

Example 6 Expression and Purification of Recombinant GST-ARTS-1Polypeptide

[0362] The cDNA sequence of the ARTS-1 extracellular domain (i.e., theectodomain; amino acids 30-941) was PCR amplified from the pTargetplasmid containing the full length ARTS-1 coding sequence. The ARTS-1extracellular domain was then cloned into the pGEX-6P-1 plasmid(Amersham/Pharmacia) and used to transform the BL21 E. coli host strain.Colonies expressing GST-ARTS-1 fusion protein were selected by Westernblotting utilizing an anti-GST antibody (Amersham/Pharmacia). Positiveclones were grown at room temperature, stimulated for 4 hours with 0.6mM isopropyl β-D-thiogalactoside (IPTG) and subsequently lysed withB-PER protein extraction reagent (Pierce). Cells were centrifuged at27,000×g for 15 minutes to separate the soluble from the insolublefractions. Following treatment with 0.2 mg/ml lysozyme for 5 minutes,the GST-ARTS-1 fusion protein was isolated from the insoluble fractionby denaturation with 6M urea in PBS. The GST-ARTS-1 fusion protein wasrefolded by serial dialysis against PBS baths containing decreasing ureaconcentrations (5 M to 0 M). The GST-ARTS-1 fusion protein was purifiedutilizing a glutathione sepharose 4B affinity column and eluted withreduced glutathione buffer using techniques known in the art.

[0363] To assess the purity of the eluted recombinant GST-ARTS-1 fusionprotein, samples were subjected to SDS-PAGE on a 4%-12% gradient gel(Novex) and stained with Coomassie brilliant blue. FIG. 6A shows theresult of this experiment. In this Figure, soluble and insoluble proteinfractions from BL21 E. coli transformed with empty pGEX-6P-1 vector(lanes 1 and 2) or the pGEX-6P-1-ARTS-1 vector (lanes 3 and 4) areshown. The GST-ARTS-1 fusion protein following elution is shown in lane5 as a predominant 132 kDa band, corresponding to the predictedmolecular weight of a GST-ARTS-1 fusion protein (104 kDa ARTS-1extracellular domain plus 26 kDa GST tag). The control purified GST tagis revealed as a predicted 26 kDa band in lane 6.

[0364] Purified recombinant GST-ARTS-1 fusion protein samples werefurther subjected to FPLC analysis (LKB LCC-500 plus,Amersham/Pharmacia) utilizing a Superose 6 HR 10/30 gel filtrationcolumn (Amersham/Pharmacia). Recombinant purified GST-ARTS-1 fusionprotein samples were eluted with PBS at a flow rate of 0.5 ml/min andfractions were collected every 1 minute. Absorbance was recorded at 280mn with a chart speed of 0.25 cm/min. FIG. 6B shows the result of thisanalysis. In this figure, FPLC analysis of the purified GST-ARTS-1revealed a single major protein elution peak which eluted atapproximately 40 minutes.

[0365] Using the aminopeptidase activity assay described below, FPLCfractions were assessed for aminopeptidase activity utilizing aphenylalanine p-nitroaniline substrate. As shown in FIG. 6C, the singlemajor protein peak revealed by FPLC analysis coeluted with a peak ofaminopeptidase activity against a phenylalanine p-nitroaniline substratein pooled fractions from 38-44 minutes.

Example 7 ARTS-1 Aminopeptidase Activity Assay

[0366] Aminopeptidase activity of the recombinant GST-ARTS-1 fusionprotein was assessed by determination of the rate of amide bondhydrolysis of amino acid p-nitroanilide substrates (Bachem). Amino acidp-nitroanilides (final concentrations 0.25 to 8 mM) were incubated atroom temperature with 24 pmoles of GST-ARTS-1 fusion protein in 200 μlof 50 mM Tris, pH 7.5 for 1 hour. Reactions were terminated by additionof 280 μl of 3M sodium acetate (pH 5.2). The rate of amide bondhydrolysis was determined by measuring the absorbance of p-nitroanilineat 380 nm (See, Table 3, supra). Spontaneous hydrolysis of the substratewas corrected for by subtracting the absorbance of control incubationswhich were terminated immediately. Kinetic constants were determined byLineweaver-Burk analysis. Each experimental point was assayed intriplicate and each determination utilized six concentrations of eachamino acid p-nitroanilide substrate. Correlation coefficients for eachline generated were greater than 0.997.

[0367] As shown in Table 3, recombinant GST-ARTS-1 protein possessedselective aminopeptidase activity against non-polar amino acidsubstrates with a four-fold range of enzyme activity. Isoleucine-pNA wasfound to be the most favorable amino acid substrate based uponk_(cat)/K_(m) determination, followed byPhe>Gly>Cys>Leu>Met>Ala>Pro>Val. Recombinant GST-ARTS-1 had no activityagainst either acidic (Asp or Glu) or basic (Arg, His, or Lys) aminoacid substrates.

Example 8 ARTS-1 Endopeptidase Activity Assay

[0368] The endopeptidase activity of recombinant GST-ARTS-1 fusionprotein was also assessed. In this assay, 5 μg of recombinant GST-ARTS-1fusion protein was incubated with 10 μg of either bovine serum albumin,human albumin, rabbit myosin heavy chain or transferrin overnight.Sample were then subjected to SDS-PAGE utilizing 4 to 12% gradient gels(Novex) and stained with Coomassie brilliant blue. In this assay,recombinant GST-ARTS-1 was shown to have no demonstrable endopeptidaseactivity against bovine serum albumin, human albumin, rabbit myosinheavy chain or human transferrin.

Example 9 Construction of Stably Transfected Cell Lines Expressing Senseand Antisense ARTS-1 cDNA's

[0369] In light of the ability of the ARTS-1 polypeptide to bind to theTNFR1 ectodomain in the yeast two-hybrid interaction assay, thefollowing experiment was conducted in order to determine whether ARTS-1has the ability promote the cleavage and shedding of the TNFR1ectodomain from the surface of human cells in culture. This experimentwas done in two phases. The first phase involved construction of stablytransfected cell lines which expressed either reduced or elevated levelsof ARTS-1 polypeptide. The NCI-H292 cell line was stably transfectedwith one of three constructs, all based on the pTarget vector (Promega).Transfection was by the Fugene system (Roche). The pTarget vectorexpresses a gene product (encoded by the neo gene) which impartsresistance to the antibiotic G-418, which kills both prokaryotic andeukaryotic cells. The construct also contains a constitutively activeCMV promoter which will express cloned DNA inserts in mammalian cells.The pTarget vectors used to transfect the NCI-H292 cells were:

[0370] 1) an empty pTarget vector,

[0371] 2) pTarget vector containing the full length ARTS-1 cDNA codingregion in the sense orientation,

[0372] 3) pTarget vector containing ARTS-1 cDNA bases 61 to 213 in theanti-sense orientation. This region overlaps the putative transcriptionstart site and intracellular and transmembrane domains. Following thetransfection of these constructs into the host cells, the transfectantswere cultured under selective pressure in RPMI-1640 media supplementedwith 10% heat-inactivated fetal calf serum and 1× antibiotic-antimycotic(Biofluids) and 500 μg/ml of the G-418 (Promega). Two independent clonesof stable transfectants containing either sense or anti-sense ARTS-1plasmid were then generated via limiting dilutions. Both sense andanti-sense clones were screened by immunoblotting (described above)utilizing anti-ARTS-1 polyclonal serum to select duplicate clones forsubsequent analysis.

[0373] Cell lines were then selected based upon enhanced or suppressedARTS-1 protein expression as determined by the immunoblotting of cellmembrane fractions, as shown in FIG. 7. Integration of the empty pTargetvector (indicated “Mock”) had little effect on endogenous ARTS-1expression compared to cells that do not contain any stably integratedplasmid (WT). The two cell lines expressing the full length ARTS-1 cDNAin the sense orientation (ARTS-1) showed a significant increase inARTS-1 protein expression, while the two cell lines expressing ARTS-1antisense sequence (AS) showed significant reduction in ARTS-1 proteinexpression. These stably integrated cell lines were examined for sTNFR1shedding activity (See, Example 11, below).

Example 10 Construction of Stably Transfected Cell Lines ExpressingMutant ARTS-1 cDNA's

[0374] The predicted ARTS-1 polypeptide contains a peptidase/proteaseconsensus motif found in the aminopeptidase family of gluzincin zincmetalloproteases. It was determined if this peptidase/protease catalyticmotif was necessary for the ability of ARTS-1 to promote shedding of theTNFR1 ectodomain. To conduct this experiment, a series of ARTS-1 mutantswere constructed which contain point mutations predicted to abolish theARTS-1 peptidase/protease activity (Devault et al., FEBS Lett.,23154-23158 [1988]; Devault et al., J. Biol. Chem., 263:4033-4040[1988]; Vallee and Auld, FEBS Lett., 257:138-140 [1989]; Vallee andAuld, Biochemistry 29:5647-5659 [1990]; Wang and Cooper, Proc. Natl.Acad. Sci. USA 90:1222-1226 [1993]). These mutations lie within the zincmetalloprotease family zinc-binding/catalytic domain consensusHEXXH(Y)₁₈E (SEQ ID NO:10; consisting of a zinc binding and catalyticsite domains). In the ARTS-1 polypeptide, this motif is located atH³⁵³ELAH(Y)₁₈E³⁷⁶ (SEQ ID NO:11). The mutations made were H353P, E354V,H353P and E354V in combination, and H357V. Mutagenesis of the ARTS-1gene open reading frame was performed using a QuikChange Site-DirectedMutagenesis Kit (Stratagene) according to the manufacturer'sinstructions.

[0375] The NCI-H292 cell line was stably transfected with one of sixconstructs, all based on the pTarget expression vector. The method usedto produce stably transfected cell lines containing these constructs isthe same as that provided in Example 9. These constructs (and resultingcell lines) were:

[0376] 1) an empty pTarget vector,

[0377] 2) the ARTS-1 cDNA (WT) coding region,

[0378] 3) the ARTS-1 cDNA encoding a H353P mutation,

[0379] 4) the ARTS-1 cDNA encoding a E354V mutation,

[0380] 5) the ARTS-1 cDNA encoding a H353P and E354V double mutation,and

[0381] 6) the ARTS-1 cDNA encoding a H357V mutation.

Example 11 TNFR1 Ectodomain Shedding Assay

[0382] The amount of TNFR1 ectodomain shedding occurring in each of thestably transfected cell lines described in Example 9 was assessed. Thelevels of sTNFR1 ectodomain in cell culture supernatants from these celllines were assayed by a commercially available sandwich-enzyme-linkedimmunosorbent assay (ELISA) technique (R & D Systems) with a lower limitof detection of 7.8 pg/ml. The protocol used was according to themanufacturer's instructions. The results of this assay are depicted inFIG. 8 as the mean of 5 independent experiments, with accompanying SEM(standard error of the mean) indicated above the bar. As can be seen inFIG. 8, the cell lines with increased ARTS-1 protein expression alsoshowed a significant increase in the amount of sTNFR1 present in cellculture supernatants as compared to cells transfected with the emptypTarget vector (Mock) or nontransfected (WT) controls. This change insTNFR1 concentration represents an approximately 200% increase in sTNFR1shedding resulting from overexpression of the ARTS-1 protein.

[0383] Conversely, cell lines showing decreased ARTS-1 proteinexpression showed significantly decreased levels of sTNFR1 in cellculture supernatants as compared to cells transfected with the emptypTarget vector, as shown in FIG. 8. This change in concentrationrepresents an approximately 80% decrease in sTNFR1 shedding resultingfrom expression of an ARTS-1 anti-sense transcript encompassing theputative translation start site.

[0384] A similar experiment analyzing the ability of ARTS-1overexpression to potentiate the cleavage and shedding of TNFRectodomain from the surface of NCI-H292 cells in response to PMAstimulation using these same cell lines is shown in FIG. 9. Cell linesoverexpressing full length ARTS-1 mRNA were stimulated with 0.1 μMphorbol 12-myristate 13-acetate (PMA), which has previously been shownto upregulate sTNFR1 shedding in NCI-H292 cells (Levine et al., Am. J.Respir. Cell Mol. Biol., 14:254-261 [1996]). As shown in FIG. 9, thecell line containing only the empty pTarget vector showed only a modestincrease in sTNFR1 shedding following 24 hours of PMA treatment,increasing from approximately 300 pg/ml to 415.3±4.5 pg/ml. However, thecell line overexpressing the ARTS-1 cDNA showed a more dramatic increasein sTNFR1 shedding following 24 hours of PMA treatment, increasing from485±16.9 pg/ml to 914.2±9.5 pg/ml

[0385] This assay was further used to measure the sTNFR1 ectodomainshedding activity of ARTS-1 peptidase/protease catalytic mutants. Theconstruction of these mutations (and resulting mutant cell lines) aredescribed in Example 10. The ability of theses mutants to regulatesTNFR1 shedding was ascertained by measuring the concentration of sTNFR1in the cell culture supernatant as described above in this Example.These results are depicted in FIG. 10 as the mean of five independentexperiments, with accompanying SEM (standard error of the mean).

[0386] As shown in FIG. 10, the cell line overexpressing the ARTS-1 cDNA(ARTS-1) showed a significantly elevated level of sTNFR1 in the culturesupernatant compared to control cell lines containing no integrated DNA(WT) or containing the empty pTarget vector (MOCK). Unexpectedly, eachof the cell lines containing mutant forms of the ARTS-1 polypeptideshowed elevated levels of sTNFR1 compared to the control lines (i.e., WTand MOCK). This experiment demonstrates the unexpected property wherethe peptidase/protease activity of the ARTS-1 polypeptide is notrequired for the sTNFR1 shedding regulatory activity of ARTS-1polypeptide.

[0387] Although not described here, IL-1 and IL-6 receptor ectodomainshedding can also be measured by ELISA-based assays using commerciallyavailable kits that measure soluble forms of the IL-1 and IL-6 receptors(R & D Systems, Catalog numbers DR1B00 and DR600, respectively). TheseELISA-based assays measure the concentration of sIL-1RII and sIL-6R,both with a lower limit of detection of 31 pg/ml.

Example 12 Effect of ARTS-1 Expression on Membrane-Associated TNFR1

[0388] The degree of TNFR1 ectodomain shedding as a function of ARTS-1protein expression was also indirectly assessed by determining therelative amounts of membrane-bound TNFR1 fragment in each of the stablytransfected cell lines described in Example 9 using Westernimmunoblotting.

[0389] Crude membrane fractions from the stably transfected NCI-H292cells described in Example 9 were prepared and protein concentrationswere quantitated, as described above. Samples from thesemembrane-derived protein preparations were resolved by SDS-PAGE andanalyzed by Western immunoblotting as described above in Example 5 usinga murine anti-human TNFR1 monoclonal primary antibody which detected themembrane fragment of the receptor (R & D System). This Western blot isshown in FIG. 11. Two independent strains of each cell line wereanalyzed in parallel.

[0390] As shown in FIG. 11, cell lines over-expressing ARTS-1 (ARTS-1)demonstrated a decrease in membrane-associated TNFR1 relative tonon-transfected (WT) or control transfected (Mock) cell lines,consistent with an increase in constitutive TNFR1 ectodomain shedding.Conversely, cell lines expressing anti-sense ARTS-1 mRNA (AS)demonstrated an increase in membrane-associated TNFR1 relative tonon-transfected (WT) or control transfected (Mock) cell lines,consistent with a reduction in constitutive TNFR1 ectodomain shedding.

Example 13 ARTS-1/TNFR1 in vivo Co-Immunoprecipitation Assays

[0391] The ability of ARTS-1 to directly interact with TNFR1 ectodomainwas assessed in vivo using a co-immunoprecipitation assay. This assayutilized anti-ARTS-1 antiserum and monoclonal anti-TNFR1 antibodies. Theimmunoprecipitated proteins were visualized by Western immunoblotting.

[0392] Crude membrane fractions from cultured NCI-H292 cells wereprepared and protein concentrations were quantitated as described above.From these membrane-derived protein preparations, 200 μg samples wereincubated with 20 μg of murine anti-human TNFR1 monoclonal antibody (R &D System) or 1 ml of anti-ARTS-1 antiserum overnight at 4° C. inimmunoprecipitation buffer (50 mM Tris-HCl, 120 mM NaCl, 0.1% TritonX-100 and COMPLETE™ protease inhibitor (Roche), pH 7.2). Following theincubation, the resulting antibody complexes were immunoprecipitated bybinding to 200 μl of immobilized protein A/G beads (Pierce) for 2 hoursat room temperature. Proteins contained in the samples were thenresolved by SDS-PAGE and analyzed by Western immunoblotting as describedabove.

[0393] Two different combinations of precipitation and immunoblottingantibody were used. The results of these immunoprecipitation experimentsare shown in FIG. 12. In one experiment (FIG. 12, top panel), theanti-TNFR1 antibody was used in the immunoprecipitation (indicated as“IP” in the Figure), and the anti-ARTS-1 antiserum was used as theprimary antibody in the immunoblotting (indicated as “IB” in theFigure). In a second experiment (FIG. 12, bottom panel), the antibodieswere reversed, where the anti-ARTS-1 antiserum was used in theimmunoprecipitation, while the anti-TNFR1 antibody was used as theprimary antibody in the immunoblotting. Also in these experiments,anti-ARTS-1 pre-immune serum (written “PI”) and a purified murine IgG1isotype (written “IgG1”) were used as negative controls.

[0394] As shown in the top panel of FIG. 12, immunoprecipitation of theNCI-H292 cell membrane proteins with an anti-TNFR1 monoclonal antibodyresulted in the coprecipitation of the 100 kDa ARTS-1 species and,conversely, as seen in the bottom panel, immunoprecipitation withanti-ARTS-1 antiserum coprecipitated the 55 kDa TNFR1. These resultsindicate an in vivo protein-protein interaction between ARTS-1 and TNFR1proteins.

[0395] Similar immunoprecipitation experiments were also performed usingthe stably-transfected NCI-H292 cell lines described in Example 9. Inthese experiments, the anti-TNFR1 antibody was used to immunoprecipitateprotein from the various membrane protein fractions, and the resultingimmunoprecipitate was examined by Western immunoblotting usinganti-ARTS-1 antiserum as the primary antibody. As shown in FIG. 13,immunoprecipitation using an anti-TNFR1 monoclonal antibody of cellmembrane protein derived from the anti-sense ARTS-1 cell line (AS)showed decreased amounts of ARTS-1 protein as compared tocontrol-transfected (Mock) or non-transfected (WT) cells, consistentwith decreased ARTS-1 protein expression in anti-sense ARTS-1 cells. Noincrease in ARTS-1 protein levels relative to control cell lines wasdetected following immunoprecipitation of ARTS-1 overexpressing celllines with an anti-TNFR1 monoclonal antibody, which likely reflectsincreased TNFR1 shedding related to ARTS-1 over-expression.

Example 14 Methods for Drug Screening

[0396] The present invention provides two examples of methods for drugscreening. These methods identify test compounds which are able toregulate ARTS-1 protein expression in a tissue, or regulate sTNFR1shedding in a cell culture system.

[0397] In the first method, cultured human NCI-H292 pulmonarymucoepidermoid carcinoma cells were exposed to a test compound, in thiscase, 4b-phorbol 12-myristate 13-acetate (PMA) at a concentration of 0.1μM. Cells were harvested and the membrane protein fraction isolated atintervals from prior to exposure to the test compound (Time=0) to 24hours following exposure to the compound. The protein samples wereanalyzed in a Western immunoblot using the anti-ARTS-1 antiserum at theprimary detection antibody, as described in Examples 4 and 5. Theprotein fractions were analyzed in duplicate, and the relativedensitometry units for each lane are shown beneath the columns. Eachimmunoblot is representative of 3 independent experiments. The resultsof this screening are shown in FIG. 14, Panel A. As can be seen in theFigure, treatment of the cultured cells resulted in an increase inARTS-1 protein expression over the course of 24 hours. Thus, thisscreening method identified a compound that is a candidate for furtherdrug development.

[0398] Results from a second screening method are illustrated in FIG.14, Panel B. In this screening method, cultured human NCI-H292 pulmonarymucoepidermoid carcinoma cells were again exposed to a test compound,PMA, at a concentration of 0.1 μM. Culture medium supernatant wascollected from the cultures at intervals between 0 and 24 hours posttreatment. The samples were analyzed by the TNFR1 ectodomain sheddingassay using an ELISA as described in Example 11. In this assay,anti-sTNFR1 antibody was used to quantitate the concentrations of sTNFR1in the cell culture supernatants. The results of this ELISA screeningare shown in FIG. 14, Panel B. As can be seen in this Figure, treatmentof the cultured cells resulted in an increase in sTNFR1 shedding overthe course of 24 hours compared to a control culture (n=5,*P) <0.05).Thus, this screening method also identified a compound that is acandidate for further drug development.

Example 15 Tumor Necrosis Factor Bioactivity (Cytotoxicity) Assay

[0399] The bioactivity of tumor necrosis factor (TNF) is measured by acell cytotoxicity assay utilizing the WEHI 164 clone-13 mousefibrosarcoma cell line (ATCC, CRL 1751). This cell line has been shownto be highly sensitive to the cytotoxic effects of human tumor necrosisfactor at concentrations as low as 0.1 pg/ml TNF following pretreatmentwith actinomycin D (Eskandari et al., Immunol. Invest., 19:69-79[1990]). In this assay, WEHI 164 cells are seeded into 96-wellmicrotiter plates at a density of 40,000 cells per well in RPMI-1640supplemented with penicillin (100 units/ml) (Advanced Biotechnologies),streptomycin (100 μg/ml) (Advanced Biotechnologies), L-glutamine (2 mM)(Advanced Biotechnologies), 10% heat-inactivated fetal-calf serum(Inovar), and actinomycin-D (0.5 μg/ml) (Calbiochem). Test samples(serum, plasma or any other body fluid) to be tested for TNF activityare diluted 6-fold in RPMI-1640 culture medium (AdvancedBiotechnologies), heat-inactivated at 56° C. for 30 minutes, and sterilefiltered. A volume of 50 μl of the diluted, heat-inactivated sample isadded to the microtiter plate wells containing the WEHI cells. Duplicatetest samples are incubated in the presence of polyclonal rabbitanti-human TNF antiserum (Genzyme) or control rabbit serum (LifeTechnologies). Cells are incubated for 20 hours, after which time 20 μlof a 5 mg/ml stock solution of the tetrazolium salt3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (alsocalled MTT or Thiazolyl blue; Sigma, Catalog Number M2128) in phosphatebuffered saline (PBS) is added to each well. Following an additional 4hours incubation, the plates are spun at 950×g for 10 minutes, the mediafrom each well is aspirated, and 100 μl of 0.04N HCl/2-propanol is addedto each well, and the plates are incubated overnight in the dark at roomtemperature. In the morning, an additional 100 μl of 0.04NHCl/2-propanol is added to each well and incubated for two hours in thedark at room temperature. Cell survival is then measuredcolorimetrically using a microplate reader with a 570 nm wavelength testfilter and a 630 nm reference filter. Cell survival in each test well isdetermined as a percentage of the optical density of control wells.TNF-specific killing is defined as the difference in cell killing withor without the anti-TNF antiserim and is compared with standard curvesproduced with recombinant human TNF-α (R & D Systems).

Example 15 Statistical Analysis

[0400] Data are presented as mean±standard error of the mean.Comparisons were made utilizing a paired two-tailed student's T testwith a Bonneferoni correction for multiple comparisons. A P value<0.005was considered significant.

[0401] These experiments demonstrate that expression of ARTS-1 proteindirectly correlates with and is necessary for soluble TNFR1 ectodomainshedding. Furthermore, ARTS-1 represents the first gene and proteinwhich have been identified which have the ability regulate TNTFR1ectodomain shedding. In addition, the present invention provides thenucleic acid and amino acid sequences of this gene and protein.

[0402] Furthermore, the compositions and methods of the presentinvention provide therapeutic applications for the treatment of a widevariety of disorders of the immune system which arise as a result ofimproper TNF activity.

[0403] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described methods and compositions of the presentinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in molecular biology and immunology and/or related fieldsare intended to be within the scope of the present invention.

1 11 1 4845 DNA Homo sapiens 1 gcacgagagc taggccggcg gcagtggtggtggcggcggc gcaagggtga gggcggcccc 60 agaaccccag gtaggtagag caagaagatggtgtttctgc ccctcaaatg gtcccttgca 120 accatgtcat ttctactttc ctcactgttggctctcttaa ctgtgtccac tccttcatgg 180 tgtcagagca ctgaagcatc tccaaaacgtagtgatggga caccatttcc ttggaataaa 240 atacgacttc ctgagtacgt catcccagttcattatgatc tcttgatcca tgcaaacctt 300 accacgctga ccttctgggg aaccacgaaagtagaaatca cagccagtca gcccaccagc 360 accatcatcc tgcatagtca ccacctgcagatatctaggg ccaccctcag gaagggagct 420 ggagagaggc tatcggaaga acccctgcaggtcctggaac acccccgtca ggagcaaatt 480 gcactgctgg ctcccgagcc cctccttgtcgggctcccgt acacagttgt cattcactat 540 gctggcaatc tttcggagac tttccacggattttacaaaa gcacctacag aaccaaggaa 600 ggggaactga ggatactagc atcaacacaatttgaaccca ctgcagctag aatggccttt 660 ccctgctttg atgaacctgc cttcaaagcaagtttctcaa tcaaaattag aagagagcca 720 aggcacctag ccatctccaa tatgccattggtgaaatctg tgactgttgc tgaaggactc 780 atagaagacc attttgatgt cactgtgaagatgagcacct atctggtggc cttcatcatt 840 tcagattttg agtctgtcag caagataaccaagagtggag tcaaggtttc tgtttatgct 900 gtgccagaca agataaatca agcagattatgcactggatg ctgcggtgac tcttctagaa 960 ttttatgagg attatttcag cataccgtatcccctaccca aacaagatct tgctgctatt 1020 cccgactttc agtctggtgc tatggaaaactggggactga caacatatag agaatctgct 1080 ctgttgtttg atgcagaaaa gtcttctgcatcaagtaagc ttggcatcac aatgactgtg 1140 gcccatgaac tggctcacca gtggtttgggaacctggtca ctatggaatg gtggaatgat 1200 ctttggctaa atgaaggatt tgccaaatttatggagtttg tgtctgtcag tgtgacccat 1260 cctgaactga aagttggaga ttatttctttggcaaatgtt ttgacgcaat ggaggtagat 1320 gctttaaatt cctcacaccc tgtgtctacacctgtggaaa atcctgctca gatccgggag 1380 atgtttgatg atgtttctta tgataagggagcttgtattc tgaatatgct aagggagtat 1440 cttagtgctg acgcatttaa aagtggtattgtacagtatc tccagaagca tagctataaa 1500 aatacaaaaa acgaggacct gtgggatagtatggcaagta tttgccctac agatggtgta 1560 aaagggatgg atggcttttg ctctagaagtcaacattcat cttcatcctc acattggcat 1620 caggaagggg tggatgtgaa aaccatgatgaacacttgga cactgcagaa gggttttccc 1680 ctaataacca tcacagtgag ggggaggaatgtacacatga agcaagagca ctacatgaag 1740 ggctctgacg gcgccccgga cactgggtacctgtggcatg ttccattgac attcatcacc 1800 agcaaatccg acatggtcca tcgatttttgctaaaaacaa aaacagatgt gctcatcctc 1860 ccagaagagg tggaatggat caaatttaatgtgggcatga atggctatta cattgtgcat 1920 tacgaggatg atggatggga ctctttgactggccttttaa aaggaacaca cacagcagtc 1980 agcagtaatg atcgggcgag tctcattaacaatgcatttc agctcgtcag cattgggaag 2040 ctgtccattg aaaaggcctt ggatttatccctgtacttga aacatgaaac tgaaattatg 2100 cccgtgtttc aaggtttgaa tgagctgattcctatgtata agttaatgga gaaaagagat 2160 atgaatgaag tggaaactca attcaaggccttcctcatca ggctgctaag ggacctcatt 2220 gataagcaga catggacaga cgagggctcagtctcagagc gaatgctgcg gagtcaacta 2280 ctactcctcg cctgtgtgca caactatcagccgtgcgtac agagggcaga aggctatttc 2340 agaaagtgga aggaatccaa tggaaacttgagcctgcctg tcgacgtgac cttggcagtg 2400 tttgctgtgg gggcccagag cacagaaggctgggattttc tttatagtaa atatcagttt 2460 tctttgtcca gtactgagaa aagccaaattgaatttgccc tctgcagaac ccaaaataag 2520 gaaaagcttc aatggctact agatgaaagctttaagggag ataaaataaa aactcaggag 2580 tttccacaaa ttcttacact cattggcaggaacccagtag gatacccact ggcctggcaa 2640 tttctgagga aaaactggaa caaacttgtacaaaagtttg aacttggctc atcttccata 2700 gcccacatgg taatgggtac aacaaatcaattctccacaa gaacacggct tgaagaggta 2760 aaaggattct tcagctcttt gaaagaaaatggttctcagc tccgttgtgt ccaacagaca 2820 attgaaacca ttgaagaaaa catcggttggatggataaga attttgataa aatcagagtg 2880 tggctgcaaa gtgaaaagct tgaacgtatgtaaaaattcc tcccttgcca ggttcctgtt 2940 atctctaatc accaacattt tgttgagtgtattttcaaac tagagatggc tgttttggct 3000 ccaactggag atactttttt cccttcaactcattttttga ctatccctgt gaaaagaata 3060 gctgttagtt tttcatgaat gggctatcgctaccatgtgt tttgttcatc acaggtgttg 3120 ccctgcaacg taaacccaag tgttgggttccctgccacag aagaataaag taccttattc 3180 ttctcatttt atagtttatg cttaagcacccgtgtccaaa accctgtacc ccatgtttat 3240 cattcataaa ctgtttcatc agtctcctcgaaagactctg aatagtcgac tactgaacaa 3300 tgaacacctg gatctgagac taagccggacgatgactggg ttaaagctct cccggctcac 3360 ccctccagac ccgctgccca tccctcttccttgctccatg cccaggggct gacttgtaaa 3420 ggccaagtca tcaagctttc ttgccctttggatgttggtc agtggggagc cggagagctg 3480 gagctggggt cggaggaggt agtaggtggaggtgttcttc cctgattccc ttgcgggatg 3540 cctcgggctg gcctcccctg agggttttagctccgagagg ggaccctctt ttccacacag 3600 ccttctccac ctctggattt tggtaactgctccctcctca tcccttcagg attagtggcc 3660 tcagtgggag tctggctttt actagtcctggcggacttgt ggtttctaca taatgtgctc 3720 gcacttttgc aaaaaatctt ctttttatagaaccctcctc agataattct gagtgtcatc 3780 tatttccctg actggtacag tatctcttctgaaaaagcag agtgcattca agtctgtagg 3840 aaaacccttt tcttagggag gtgattttttttctctctct gcttcttatt tggcctactt 3900 tacaatttct aactaactag ttattggcatttactgacag taaattattg cagtcaccaa 3960 taaatgatag tacattgtga aacaaaatatttgctcatat tagcaaatag gacattcttt 4020 ggctttgaag tctttctttt gtgaagacttcacacacggt tgcttcagca cacagttgct 4080 gctcaggttt tatgtataga tgataataatagaaagcaca gtttactaac atggtaaacc 4140 aacggagttc aagtcaagtc agttaataccctaagaatta gattttattt cttattctga 4200 aaacttgcta cacagggact tatctaacccatagtgtgct ctgttgctga cttgattcaa 4260 gttgcagcgt gttttgcgct gactctaaggtgcggaaatc ctcacacctg gcaaaggaga 4320 attcaaactg aactttttga atataaggcaaaaacttcaa gataagggaa tatgattgat 4380 gattggtacg aaaaatgtca aaatgtgttcccctaataca cgacaaaata gagtgacttc 4440 tggacataaa tctgccattt attaaaccattcactacaac aaataaatag gtataaaagt 4500 ggaattggaa tttttatact tatttgttgtagtgaatggt ttaataaaaa tagaaatcac 4560 tggtaatttc caccccaaac taaactatttcccttctttt aaaaaaatac acaaccaaga 4620 ttttaatgta aaatattttg ctttaattgtattttatgcc ttgattaatg aaacatggaa 4680 atattgattt tcagttttgg tcacctgaggaacctatctt tgtttgcttt tggaaaagcc 4740 cattttctaa acagatacaa tattgccacaacaatgtgca gaaacctttt tgataataaa 4800 aaattgttct ttgcctctaa aaaaaaaaaaaaaaaaaaaa aaaaa 4845 2 941 PRT Homo sapiens 2 Met Val Phe Leu Pro LeuLys Trp Ser Leu Ala Thr Met Ser Phe Leu 1 5 10 15 Leu Ser Ser Leu LeuAla Leu Leu Thr Val Ser Thr Pro Ser Trp Cys 20 25 30 Gln Ser Thr Glu AlaSer Pro Lys Arg Ser Asp Gly Thr Pro Phe Pro 35 40 45 Trp Asn Lys Ile ArgLeu Pro Glu Tyr Val Ile Pro Val His Tyr Asp 50 55 60 Leu Leu Ile His AlaAsn Leu Thr Thr Leu Thr Phe Trp Gly Thr Thr 65 70 75 80 Lys Val Glu IleThr Ala Ser Gln Pro Thr Ser Thr Ile Ile Leu His 85 90 95 Ser His His LeuGln Ile Ser Arg Ala Thr Leu Arg Lys Gly Ala Gly 100 105 110 Glu Arg LeuSer Glu Glu Pro Leu Gln Val Leu Glu His Pro Arg Gln 115 120 125 Glu GlnIle Ala Leu Leu Ala Pro Glu Pro Leu Leu Val Gly Leu Pro 130 135 140 TyrThr Val Val Ile His Tyr Ala Gly Asn Leu Ser Glu Thr Phe His 145 150 155160 Gly Phe Tyr Lys Ser Thr Tyr Arg Thr Lys Glu Gly Glu Leu Arg Ile 165170 175 Leu Ala Ser Thr Gln Phe Glu Pro Thr Ala Ala Arg Met Ala Phe Pro180 185 190 Cys Phe Asp Glu Pro Ala Phe Lys Ala Ser Phe Ser Ile Lys IleArg 195 200 205 Arg Glu Pro Arg His Leu Ala Ile Ser Asn Met Pro Leu ValLys Ser 210 215 220 Val Thr Val Ala Glu Gly Leu Ile Glu Asp His Phe AspVal Thr Val 225 230 235 240 Lys Met Ser Thr Tyr Leu Val Ala Phe Ile IleSer Asp Phe Glu Ser 245 250 255 Val Ser Lys Ile Thr Lys Ser Gly Val LysVal Ser Val Tyr Ala Val 260 265 270 Pro Asp Lys Ile Asn Gln Ala Asp TyrAla Leu Asp Ala Ala Val Thr 275 280 285 Leu Leu Glu Phe Tyr Glu Asp TyrPhe Ser Ile Pro Tyr Pro Leu Pro 290 295 300 Lys Gln Asp Leu Ala Ala IlePro Asp Phe Gln Ser Gly Ala Met Glu 305 310 315 320 Asn Trp Gly Leu ThrThr Tyr Arg Glu Ser Ala Leu Leu Phe Asp Ala 325 330 335 Glu Lys Ser SerAla Ser Ser Lys Leu Gly Ile Thr Met Thr Val Ala 340 345 350 His Glu LeuAla His Gln Trp Phe Gly Asn Leu Val Thr Met Glu Trp 355 360 365 Trp AsnAsp Leu Trp Leu Asn Glu Gly Phe Ala Lys Phe Met Glu Phe 370 375 380 ValSer Val Ser Val Thr His Pro Glu Leu Lys Val Gly Asp Tyr Phe 385 390 395400 Phe Gly Lys Cys Phe Asp Ala Met Glu Val Asp Ala Leu Asn Ser Ser 405410 415 His Pro Val Ser Thr Pro Val Glu Asn Pro Ala Gln Ile Arg Glu Met420 425 430 Phe Asp Asp Val Ser Tyr Asp Lys Gly Ala Cys Ile Leu Asn MetLeu 435 440 445 Arg Glu Tyr Leu Ser Ala Asp Ala Phe Lys Ser Gly Ile ValGln Tyr 450 455 460 Leu Gln Lys His Ser Tyr Lys Asn Thr Lys Asn Glu AspLeu Trp Asp 465 470 475 480 Ser Met Ala Ser Ile Cys Pro Thr Asp Gly ValLys Gly Met Asp Gly 485 490 495 Phe Cys Ser Arg Ser Gln His Ser Ser SerSer Ser His Trp His Gln 500 505 510 Glu Gly Val Asp Val Lys Thr Met MetAsn Thr Trp Thr Leu Gln Lys 515 520 525 Gly Phe Pro Leu Ile Thr Ile ThrVal Arg Gly Arg Asn Val His Met 530 535 540 Lys Gln Glu His Tyr Met LysGly Ser Asp Gly Ala Pro Asp Thr Gly 545 550 555 560 Tyr Leu Trp His ValPro Leu Thr Phe Ile Thr Ser Lys Ser Asp Met 565 570 575 Val His Arg PheLeu Leu Lys Thr Lys Thr Asp Val Leu Ile Leu Pro 580 585 590 Glu Glu ValGlu Trp Ile Lys Phe Asn Val Gly Met Asn Gly Tyr Tyr 595 600 605 Ile ValHis Tyr Glu Asp Asp Gly Trp Asp Ser Leu Thr Gly Leu Leu 610 615 620 LysGly Thr His Thr Ala Val Ser Ser Asn Asp Arg Ala Ser Leu Ile 625 630 635640 Asn Asn Ala Phe Gln Leu Val Ser Ile Gly Lys Leu Ser Ile Glu Lys 645650 655 Ala Leu Asp Leu Ser Leu Tyr Leu Lys His Glu Thr Glu Ile Met Pro660 665 670 Val Phe Gln Gly Leu Asn Glu Leu Ile Pro Met Tyr Lys Leu MetGlu 675 680 685 Lys Arg Asp Met Asn Glu Val Glu Thr Gln Phe Lys Ala PheLeu Ile 690 695 700 Arg Leu Leu Arg Asp Leu Ile Asp Lys Gln Thr Trp ThrAsp Glu Gly 705 710 715 720 Ser Val Ser Glu Arg Met Leu Arg Ser Gln LeuLeu Leu Leu Ala Cys 725 730 735 Val His Asn Tyr Gln Pro Cys Val Gln ArgAla Glu Gly Tyr Phe Arg 740 745 750 Lys Trp Lys Glu Ser Asn Gly Asn LeuSer Leu Pro Val Asp Val Thr 755 760 765 Leu Ala Val Phe Ala Val Gly AlaGln Ser Thr Glu Gly Trp Asp Phe 770 775 780 Leu Tyr Ser Lys Tyr Gln PheSer Leu Ser Ser Thr Glu Lys Ser Gln 785 790 795 800 Ile Glu Phe Ala LeuCys Arg Thr Gln Asn Lys Glu Lys Leu Gln Trp 805 810 815 Leu Leu Asp GluSer Phe Lys Gly Asp Lys Ile Lys Thr Gln Glu Phe 820 825 830 Pro Gln IleLeu Thr Leu Ile Gly Arg Asn Pro Val Gly Tyr Pro Leu 835 840 845 Ala TrpGln Phe Leu Arg Lys Asn Trp Asn Lys Leu Val Gln Lys Phe 850 855 860 GluLeu Gly Ser Ser Ser Ile Ala His Met Val Met Gly Thr Thr Asn 865 870 875880 Gln Phe Ser Thr Arg Thr Arg Leu Glu Glu Val Lys Gly Phe Phe Ser 885890 895 Ser Leu Lys Glu Asn Gly Ser Gln Leu Arg Cys Val Gln Gln Thr Ile900 905 910 Glu Thr Ile Glu Glu Asn Ile Gly Trp Met Asp Lys Asn Phe AspLys 915 920 925 Ile Arg Val Trp Leu Gln Ser Glu Lys Leu Glu Arg Met 930935 940 3 24 DNA Artificial Sequence Synthetic 3 ataaccatca cagtgagggggagg 24 4 24 DNA Artificial Sequence Synthetic 4 tagttgactc cgcagcattcgctc 24 5 26 DNA Artificial Sequence Synthetic 5 gcaagaagat ggtgtttctgcccctc 26 6 24 DNA Artificial Sequence Synthetic 6 ttacatacgt tcaagcttttcact 24 7 17 PRT Artificial Sequence Synthetic 7 Arg Gly Arg Asn Val HisMet Lys Gln Glu His Tyr Met Lys Gly Ser 1 5 10 15 Asp 8 12 PRTArtificial Sequence Synthetic 8 Thr Val Ala His Glu Leu Ala His Gln TrpPhe Gly 1 5 10 9 8 PRT Artificial Sequence Synthetic 9 Leu Trp Leu AsnGlu Gly Phe Ala 1 5 10 24 PRT Artificial Sequence Synthetic 10 His GluXaa Xaa His Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr 1 5 10 15 TyrTyr Tyr Tyr Tyr Tyr Tyr Glu 20 11 24 PRT Artificial SequenceArtificial/Unknown 11 His Glu Leu Ala His Tyr Tyr Tyr Tyr Tyr Tyr TyrTyr Tyr Tyr Tyr 1 5 10 15 Tyr Tyr Tyr Tyr Tyr Tyr Tyr Glu 20

What is claimed is:
 1. An isolated nucleic acid having the nucleotidesequence set forth in SEQ ID NO:1.
 2. An isolated polypeptide having theamino acid sequence set forth in SEQ ID NO:2.
 3. An isolated nucleicacid encoding the polypeptide set forth in SEQ ID NO:2.
 4. A recombinantvector comprising the nucleic acid of claim
 3. 5. A host cell comprisingthe recombinant vector of claim 4, wherein said host cell is selectedfrom the group consisting of prokaryotic host cells and eukaryotic hostcells.
 6. An antibody directed against at least a portion of saidpolypeptide of claim 2, wherein said antibody is selected from the groupconsisting of monoclonal antibodies and polyclonal antibodies.
 7. Anisolated nucleic acid substantially homologous to the nucleic acid ofclaim 1, wherein said nucleic acid is capable of hybridizing under highstringency conditions to the nucleic acid of claim
 1. 8. The isolatednucleic acid of claim 7, wherein said nucleic acid encodes a polypeptidehaving the ability to regulate the shedding of the extracellular domainof at least one cytokine receptor.
 9. The isolated nucleic acid of claim8, wherein said cytokine receptor is selected from the group consistingof type-1 tumor necrosis factor receptor, type I interleukin-1 cytokinereceptor, type II interleukin-1 cytokine receptor, and interleukin-6cytokine receptor alpha-chain gp80.
 10. A method for the isolation of anamplifiable nucleic acid substantially homologous to the nucleic acid ofclaim 1, comprising: a) providing: i) a sample comprising templatenucleic acid suspected of encoding a gene substantially homologous tothe nucleic acid of claim 1, and ii) at least two primers; b) annealingsaid primers to said template nucleic acid; c) extending said primerswith reiterated DNA synthesis under conditions such that said templatenucleic acid is amplified to produce an amplified product; and d)visualizing and isolating said amplified product.
 11. The amplifiedproduct of claim
 10. 12. The amplified product of claim 11, wherein saidamplified product encodes a polypeptide having the ability to regulatethe shedding of the extracellular domain of at least one cytokinereceptor.
 13. The amplified product of claim 12, wherein said cytokinereceptor is selected from the group consisting of type-1 tumor necrosisfactor receptor, type I interleukin-1 cytokine receptor, type IIinterleukin-1 cytokine receptor, and interleukin-6 cytokine receptoralpha-chain gp80.
 14. A method for regulating the shedding of theextracellular domain of at least one cytokine receptor, comprising thesteps of: a) providing: i) the recombinant vector of claim 4, whereinsaid vector comprises the nucleic acid of claim 3 in the senseorientation, ii) a first tissue containing one or more cells expressinga cytokine receptor, and iii) a second tissue comprising one or morecells capable of expressing the polypeptide encoded by said recombinantvector; and b) delivering said vector to said cells of said secondtissue in the presence of said first tissue, under conditions whichresult in regulation of shedding of said cytokine receptor from cells ofsaid first tissue.
 15. The method of claim 14, wherein said cytokinereceptor is selected from the group consisting of type-1 tumor necrosisfactor receptor, type I interleukin-1 cytokine receptor, type IIinterleukin-1 cytokine receptor, and interleukin-6 cytokine receptoralpha-chain gp80.
 16. The method of claim 14, wherein said deliveringsaid vector to said second tissue comprises a means of intracellulardelivery selected from the group consisting of direct nucleic acidadministration, liposome administration, viral vector delivery, and exvivo gene delivery followed by transplantation.
 17. A method forregulating the shedding of the extracellular domain of at least onecytokine receptor, comprising the steps of: a) providing: i) therecombinant vector of claim 4, wherein said recombinant vector comprisesat least a transcribeable portion of the nucleic acid of claim 1 in anantisense orientation, ii) a first tissue comprising one or more cellsexpressing a cytokine receptor, and iii) a second tissue comprising oneor more cells expressing the endogenous polypeptide of SEQ ID NO:2, andone or more cells capable of transcribing said antisense nucleic acid;and b) delivering said vector to said second tissue in the presence ofsaid first tissue, under conditions that result in regulation ofshedding of said cytokine receptor from the cells of said first tissue.18. The method of claim 17, wherein said cytokine receptor is selectedfrom the group consisting of type-1 tumor necrosis factor receptor, typeI interleukin-1 cytokine receptor, type II interleukin-1 cytokinereceptor, and interleukin-6 cytokine receptor alpha-chain gp80.
 19. Themethod of claim 17, wherein said delivering said vector to said secondtissue comprises a means of intracellular delivery selected from thegroup consisting of direct nucleic acid administration, liposomeadministration, viral vector delivery, and ex vivo gene deliveryfollowed by transplantation.
 20. A method for regulating the shedding ofthe extracellular domain of at least one cytokine receptor, comprisingthe steps of: a) providing: i) a polypeptide having the amino acidsequence set forth in SEQ ID NO:2, and ii) a tissue comprising one ormore cells expressing a cytokine receptor on their plasma membraneextracellular surface; and b) delivering said polypeptide to said tissueunder conditions such that said polypeptide regulates the shedding ofthe cytokine receptor from the surface of said cells of said tissue. 21.The method of claim 20, wherein said cytokine receptor is selected fromthe group consisting of type-1 tumor necrosis factor receptor, type Iinterleukin-1 cytokine receptor, type II interleukin-1 cytokinereceptor, and interleukin-6 cytokine receptor alpha-chain gp80.
 22. Themethod of claim 20, wherein said delivering said polypeptide to saidtissue comprises a means of delivery selected from the group consistingof oral administration, intra-arterial injection, intravenous injection,intramuscular injection, intraperitoneal injection, subcutaneousinjection, suppository, local surgical administration, systemic surgicaladministration, catheter, and any combination of these means ofdelivery.
 23. A method for regulating the shedding of the extracellulardomain of at least one cytokine receptor, comprising the steps of: a)providing: i) the antibody of claim 6, and ii) a tissue comprising oneor more cells expressing a cytokine receptor and the endogenouspolypeptide of SEQ ID NO:2; and b) delivering said antibody to saidtissue under conditions such that said antibody regulates the sheddingof said cytokine receptor from the surface of said cells of said tissue.24. The method of claim 23, wherein said cytokine receptor is selectedfrom the group consisting of type-1 tumor necrosis factor receptor, typeI interleukin-1 cytokine receptor, type II interleukin-1 cytokinereceptor, and interleukin-6 cytokine receptor alpha-chain gp80.
 25. Themethod of claim 23, wherein said means of delivery is selected from thegroup consisting of oral administration, intra-arterial injection,intravenous injection, intramuscular injection, intraperitonealinjection, subcutaneous injection, suppository, local surgicaladministration, systemic surgical administration, catheter, and anycombination of these means of delivery.
 26. A method for treating asubject, comprising the steps of: a) providing: i) a compositionselected from the group consisting of the recombinant vector of claim 4,wherein said vector contains the nucleic acid of claim 3 in the senseorientation, the recombinant vector of claim 4, wherein said vectorcontains at least a portion of said nucleic acid of claim 1 in theantisense orientation, the polypeptide of claim 2, and the antibody ofclaim 6, ii) a subject, and iii) a means of delivery of said compositionto at least one tissue of said subject; and b) delivering saidcomposition to said subject using said means of delivery.
 27. The methodof claim 26, wherein said subject is selected from the group consistingof a subject displaying pathology resulting from abnormal cytokineactivity, a subject suspected of displaying pathology resulting fromabnormal cytokine activity, and a subject at risk of displayingpathology resulting from abnormal cytokine activity.
 28. The method ofclaim 27, wherein said cytokine activity is mediated by a cytokineselected from the group consisting of tumor necrosis factor α,interleukin-1 alpha, interleukin-1 beta, and interleukin-6.
 29. Themethod of claim 26, wherein said subject is a human.
 30. The method ofclaim 26, wherein said means of delivery is selected from the groupconsisting of oral administration, intra-arterial injection, intravenousinjection, intramuscular injection, intraperitoneal injection,subcutaneous injection, suppository, local surgical administration,systemic surgical administration, catheter, and any combination of thesemeans of delivery.
 31. The method of claim 30, wherein said means ofdelivery is further selected from the group consisting of direct nucleicacid administration, liposome administration, viral vector delivery, andex vivo gene delivery followed by transplantation.
 32. A means fordetecting an ARTS-1 mRNA in a sample, wherein said means comprises atleast a portion of the nucleic acid of claim 1 complementary to at leasta portion of said ARTS-1 mRNA, and further wherein said nucleic acid isa probe.
 33. The means of claim 32, wherein said means comprisesNorthern blotting.
 34. The means of claim 32, wherein said sample is atissue sample from a subject.
 35. A means for detecting an ARTS-1polypeptide in a sample, wherein said means comprises the antibody ofclaim
 6. 36. The means of claim 35, wherein said means comprises Westernimmunoblotting.
 37. The means of claim 35, wherein said means comprisesan enzyme-linked immunosorbent assay.
 38. The means of claim 35, whereinsaid sample is a tissue sample from a subject.
 39. A diagnostic kitcomprising a means to measure ARTS-1 expression, wherein said means isselected from the group consisting of the means of claim 32 and claim35.
 40. A method for drug screening to identify drugs having the abilityto regulate ARTS-1 expression comprising the steps of: a) providing: i)a drug, ii) cultured cells, and iii) a means to measure ARTS-1expression, wherein said means is selected from the group consisting ofthe means of claim 32 and claim 35; b) exposing said cells to said drug;and c) using said means to measure ARTS-1 expression.
 41. The method ofclaim 40, wherein said cultured cells are human NCI-H292 pulmonarymucoepidermoid carcinoma cells.
 42. A method for drug screening toidentify drugs capable of regulating the peptidase activity of ARTS-1,comprising the steps of: a) providing: i) purified ARTS-1 polypeptide,ii) an amino acid p-nitroaniline, iii) a means to measure amino acidp-nitroaniline cleavage, and iv) a drug; b) exposing said purifiedARTS-1 polypeptide to said amino acid p-nitroaniline in the absence andpresence of said drug; and c) measuring amino acid p-nitroanilinecleavage in the absence and presence of said drug.
 43. The method ofclaim 42, wherein said purified ARTS-1 polypeptide comprisesglutathione-S-transferase.
 44. The method of claim 42, wherein saidamino acid p-nitroaniline is selected from the group consisting ofisoleucine p-nitroanilide, phenylalanine p-nitroanilide and glycinep-nitroanilide.
 45. The method of claim 42, wherein said means tomeasure amino acid p-nitroaniline cleavage comprises measuringabsorbance at 380 nm.
 46. A method for drug screening to identify drugscapable of regulating the shedding of a cytokine receptor, comprisingthe steps of: a) providing: i) cultured cells expressing a cytokinereceptor, ii) a means to quantitate the concentration of the solubleform of said cytokine receptor in the supernatants of said culturedcells, and iii) a drug; b) culturing said cells in the absence andpresence of said drug; c) quantitating the concentration of said solubleform of said cytokine receptor in said supernatants of said culturedcells using said means; and d) comparing the concentrations of saidsoluble cytokine receptor in said supernatants of said cell cultures inthe absence and presence of drug.
 47. The method of claim 46, whereinsaid cytokine receptor is selected from the group consisting of type-1tumor necrosis factor receptor, type II interleukin-1 cytokine receptor,and interleukin-6 cytokine receptor alpha-chain gp80.
 48. The method ofclaim 46, wherein said means to quantitate the concentration of thesoluble form of a cytokine receptor is an enzyme-linked immunosorbentassay.
 49. The method of claim 46, wherein said cultured cells arecultured human NCI-H292 pulmonary mucoepidermoid carcinoma cells.